Methods for spatial analysis using rna-templated ligation

ABSTRACT

Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample using RNA-templated ligation.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119(e), this application is a continuation ofInternational Application PCT/US2020/066720, with an internationalfiling date of Dec. 22, 2020, which claims the benefit to U.S.Provisional Patent Application No. 62/952,736, filed Dec. 23, 2019; U.S.Provisional Patent Application No. 62/969,458, filed Feb. 3, 2020; U.S.Provisional Patent Application No. 63/087,061, filed Oct. 2, 2020; andU.S. Provisional Patent Application No. 63/108,088, filed Oct. 30, 2020.The contents of each of these applications are incorporated herein byreference in their entireties.

BACKGROUND

Cells within a tissue have differences in cell morphology and/orfunction due to varied analyte levels (e.g., gene and/or proteinexpression) within the different cells. The specific position of a cellwithin a tissue (e.g., the cell's position relative to neighboring cellsor the cell's position relative to the tissue microenvironment) canaffect, e.g., the cell's morphology, differentiation, fate, viability,proliferation, behavior, signaling, and cross-talk with other cells inthe tissue.

Spatial heterogeneity has been previously studied using techniques thattypically provide data for a handful of analytes in the context ofintact tissue or a portion of a tissue (e.g., tissue section), orprovide significant analyte data from individual, single cells, butfails to provide information regarding the position of the single cellsfrom the originating biological sample (e.g., tissue).

Generally, targeting a particular analyte in a biological sampleutilizes a capture probe that targets a common transcript sequence suchas a poly(A) mRNA-like tail. However, this approach is capable ofdetecting a high number of off target analytes. Methods such asRNA-templated ligation offer an alternative to indiscriminant capture ofa common transcript sequence. See, e.g., Yeakley, PLoS One, 25;12(5):e0178302 (2017), which is incorporated by reference in itsentirety. However, there remains a need to develop an alternative tocommon transcript sequence (e.g., poly(A) mRNA-like tail) capture oftarget analytes that is capable of detecting an analyte(s) in an entiretranscriptome while providing information regarding the spatial locationand abundance of a target analyte.

SUMMARY

Targeted RNA capture is an attractive alternative to poly(A) mRNAcapture in order to interrogate spatial gene expression in a sample(e.g., an FFPE tissue). Compared to poly(A) mRNA capture, targeted RNAcapture as described herein is less affected by RNA degradationassociated with FFPE fixation compared to methods dependent on oligo-dTcapture and reverse transcription of mRNA. Further targeted RNA captureas described herein allows for sensitive measurement of specific genesof interest that otherwise might be missed with a whole transcriptomicapproach. Targeted RNA capture can be used to capture a defined set ofRNA molecules of interest, or it can be used at a whole transcriptomelevel, or anything in between. When combined with the spatial methodsdisclosed herein, the location and abundance of the RNA targets can bedetermined.

In one aspect, this disclosure features a method for determining alocation of an analyte in a biological sample including: (a) providingthe biological sample on an array including a plurality of captureprobes, where a capture probe of the plurality includes: (i) a spatialbarcode and (ii) a capture domain; (b) contacting a first probe and asecond probe with the biological sample, where the first probe and thesecond probe each include one or more sequences that are substantiallycomplementary to sequences of the analyte, and where the second probeincludes a capture probe capture domain; (c) hybridizing the first probeand the second probe to the analyte; (d) generating a ligation productby ligating the first probe and the second probe; (e) releasing theligated product from the analyte; (f) hybridizing the ligation productto the capture domain; and (g) determining (i) all or a part of thesequence of the ligation product bound to the capture domain, or acomplement thereof, and (ii) all or a part of the sequence of thespatial barcode, or a complement thereof, and using the determinedsequence of (i) and (ii) to identify the location of the analyte in thebiological sample. In some instances, the methods include determiningthe abundance and location of the analyte in the biological sample. Insome instances, the methods include determining the abundance of ananalyte at a location in the biological sample.

In some embodiments, the first probe and the second probe aresubstantially complementary to adjacent sequences of the analyte. Insome embodiments, the first probe and the second probe hybridize tosequences that are not adjacent to each other on the analyte.

In some embodiments, the method further includes hybridizing a thirdprobe to the first probe and the second probe, where the third probeincludes a first sequence that is substantially complementary to aportion of the first probe and a second sequence that is substantiallycomplementary to a portion of the second probe. In some embodiments,hybridizing the first probe and the second probe to the analyte isperformed at a first temperature. In some embodiments, hybridizing thethird probe to the first probe and the second probe is performed at asecond temperature. In some embodiments, the first temperature is ahigher temperature than the second temperature. In some embodiments, thefirst temperature is from about 50° C. to about 75° C., from about 55°C. to about 70° C., or from about 60° C. to about 65° C. In someembodiments, the second temperature is from about 15° C. to about 35°C., from about 20° C. to about 30° C., or from about 25° C. to about 30°C. In some embodiments, the first probe is extended with a DNApolymerase, thereby filling in a gap between the first probe and thesecond probe and generating an extended first probe.

In some embodiments, the first probe includes a sequence that issubstantially complementary to a first target sequence of the analyte.In some embodiments, the second probe further includes: (i) a firstsequence that is substantially complementary to a second target sequenceof the analyte; (ii) a linker sequence; (iii) a second sequence that issubstantially complementary to a third target sequence of the analyte;and (iv) a capture probe capture domain that is capable of binding to acapture domain of a capture probe. In some embodiments, the first targetsequence of the analyte is directly adjacent to the second targetsequence of the analyte. In some embodiments, the second target sequenceis not directly adjacent to the third target sequence on the analyte. Insome embodiments, the second target sequence and the third targetsequence are (i) on different exons of the analyte or (ii) locatedwithin the same exon of the analyte but are not adjacent on the analyte.

In some embodiments, the second probe includes a sequence that issubstantially complementary to a third target sequence of the analyte.In some embodiments, the first probe includes: (i) a first sequence thatis substantially complementary to a first target sequence of theanalyte; (ii) a linker sequence; and (iii) second sequence that issubstantially complementary to second target sequence of the analyte. Insome embodiments, the second target sequence is directly adjacent to thethird target sequence. In some embodiments, the first target sequence isnot directly adjacent to the second target sequence on the analyte. Insome embodiments, the first target sequence and second target sequenceare (i) on different exons of the analyte or (ii) located within thesame exon but are not directly adjacent on the analyte.

In some embodiments, the linker sequence includes a total of about 1nucleotide to about 100 nucleotides. In some embodiments, the linkerfurther includes a barcode sequence that serves as a proxy foridentifying the analyte.

In some embodiments, the first probe includes at least two ribonucleicacid bases at the 3′ end, and where the second probe includes aphosphorylated nucleotide at the 5′ end.

In some embodiments, generating a ligation product includes ligating (i)the first probe to the second probe or (ii) the extended first probe tothe second probe using enzymatic ligation or chemical ligation, wherethe enzymatic ligation utilizes a ligase. In some embodiments, theligase is one or more of a T4 RNA ligase (Rnl2), a splintR ligase, asingle stranded DNA ligase, or a T4 DNA ligase.

In some embodiments, the second probe includes a pre-adenylatedphosphate group at its 5′ end, and where the first probe includes atleast two ribonucleic acid bases at the 3′ end. In some embodiments, thestep of generating a ligation product includes ligating a 3′ end of thefirst probe to the 5′ end of the second probe using a ligase that doesnot require adenosine triphosphate for ligase activity. In someembodiments, the ligase is selected from the group consisting of:thermostable 5′ AppDNA/RNA Ligase, truncated T4 RNA Ligase 2, truncatedT4 RNA Ligase 2 K227Q, truncated T4 RNA Ligase 2 KQ, Chlorella VirusPBCV-1 DNA Ligase, or any combination thereof.

In some embodiments, the first probe further includes a functionalsequence, where the functional sequence is a primer sequence.

In some embodiments, the method further includes providing a captureprobe capture domain blocking moiety that interacts with the captureprobe capture domain. In some embodiments, the method further includesreleasing the capture probe capture domain blocking moiety from thecapture probe capture domain prior to step (f). In some embodiments, thecapture probe capture domain includes a poly-adenylated (poly(A))sequence or a complement thereof. In some embodiments, the capture probecapture domain blocking moiety includes a poly-uridine sequence, apoly-thymidine sequence, or both. In some embodiments, releasing thepoly-uridine sequence from the poly(A) sequence includes denaturing theligation product or contacting the ligation product with anendonuclease, exonuclease or ribonuclease. In some embodiments, thecapture probe capture domain includes a sequence that is complementaryto all or a portion of the capture domain of the capture probe. In someembodiments, the capture probe capture domain includes a degeneratesequence.

In some embodiments, the first probe and/or the second probe is a DNAprobe.

In some embodiments, the third probe is a DNA probe.

In some embodiments, the capture probe capture domain blocking moiety isa DNA probe.

In some embodiments, the releasing step (f) includes removing theligated probe from the analyte.

In some embodiments, the releasing of (i) the ligation product from theanalyte or (ii) the capture probe capture domain blocking moiety fromthe capture domain binding domain, includes contacting the ligated probewith an endoribonuclease. In some embodiments, the endoribonuclease isone or more of RNase H, RNase A, RNase C, or RNase I. In someembodiments, the RNase H includes RNase H1, RNase H2, or RNase H1 andRNase H2.

In some embodiments, the biological sample is a tissue sample. In someembodiments, the tissue sample is a formalin-fixed, paraffin-embedded(FFPE) tissue sample, a fresh or a frozen tissue sample. In someembodiments, the tissue sample is the FFPE tissue sample, and the tissuesample is decrosslinked. In some embodiments, the biological sample waspreviously stained. In some embodiments, the biological sample waspreviously stained using immunofluorescence or immunohistochemistry. Insome embodiments, the biological sample was previously stained usinghematoxylin and eosin.

In some embodiments, the method further includes contacting thebiological sample with a permeabilization agent, where thepermeabilization agent is selected from an organic solvent, a detergent,and an enzyme, or a combination thereof. In some embodiments, thepermeabilization agent is selected from the group consisting of: anendopeptidase, a protease sodium dodecyl sulfate (SDS), polyethyleneglycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20,N-lauroylsarcosine sodium salt solution, saponin, Triton X-100™, andTween-20™. In some embodiments, the endopeptidase is pepsin orproteinase K.

In some embodiments, the method further includes, prior to step (a),fixing the biological sample. In some embodiments, the step of fixingthe biological sample is performed using one or both of methanol andacetone.

In some embodiments, the analyte includes RNA. In some embodiments, theRNA is an mRNA.

In some embodiments, the determining step includes amplifying all orpart of the ligation product specifically bound to the capture domain.In some embodiments, an amplifying product includes (i) all or part ofsequence of the ligation product specifically bound to the capturedomain, or a complement thereof, and (ii) all or a part of the sequenceof the spatial barcode, or a complement thereof. In some embodiments,the determining step includes sequencing. In some embodiments, thesequencing step includes in situ sequencing, Sanger sequencing methods,next-generation sequencing methods, and nanopore sequencing.

In another aspect, this disclosure features a kit including: (a) asubstrate including a plurality of capture probes including a spatialbarcode and a capture domain; (b) a system including: a plurality offirst probes and second probes, where a first probe and a second probeeach includes sequences that are substantially complementary to ananalyte, and where the second probe includes a capture binding domain;and (c) instructions for performing the method of any one of thepreceding claims.

In another aspect, this disclosure features a kit including: (a) anarray including a plurality of capture probes; (b) a plurality of probesincluding a first probe and a second, where the first probe and thesecond probe are substantially complementary to adjacent sequences of ananalyte, where the second probe includes (i) a capture probe capturedomain that is capable of binding to a capture domain of the captureprobe and (ii) a linker sequence; (c) a plurality of enzymes including aribonuclease and a ligase; and (d) instructions for performing themethod of any one of the preceding claims.

In another aspect, this disclosure features a kit including: (a) anarray including a plurality of capture probes; (b) a plurality of probesincluding a first probe and a second probe, where the first probe andthe second probe are substantially complementary to adjacent sequencesof an analyte, where the first probe includes a linker sequence, wherethe second probe includes a capture probe capture domain that is capableof binding to a capture domain of the capture probe; (c) a plurality ofenzymes including a ribonuclease and a ligase; and (d) instructions forperforming the method of any one of the preceding claims.

In some embodiments, the kit includes a second probe including apreadenylated phosphate group at its 5′ end, and a ligase that does notrequire adenosine triphosphate for ligase activity.

In another aspect, this disclosure features a composition including aspatial array including capture probes, where the capture probes includea spatial barcode and a capture domain, a biological sample on thespatial array where the biological sample includes a plurality ofanalytes of interest, a first probe oligonucleotide and a second probeoligonucleotide hybridized to an analyte and ligated together, where thefirst probe oligonucleotide and the second probe oligonucleotide eachinclude a sequence that is substantially complementary to adjacentsequences of the analyte and where one of the first probe or the secondprobe includes a capture probe capture domain.

In some embodiments, the composition further includes an RNase H enzyme.In some embodiments, the composition further includes a ligase. In someembodiments, the probe oligonucleotide that does not include a captureprobe capture domain includes a functional domain.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, patent application, or item ofinformation was specifically and individually indicated to beincorporated by reference. To the extent publications, patents, patentapplications, and items of information incorporated by referencecontradict the disclosure contained in the specification, thespecification is intended to supersede and/or take precedence over anysuch contradictory material.

Where values are described in terms of ranges, it should be understoodthat the description includes the disclosure of all possible sub-rangeswithin such ranges, as well as specific numerical values that fallwithin such ranges irrespective of whether a specific numerical value orspecific sub-range is expressly stated.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection but does notnecessarily refer to every item in the collection, unless expresslystated otherwise, or unless the context of the usage clearly indicatesotherwise.

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “a cell”includes one or more cells, comprising mixtures thereof. “A and/or B” isused herein to include all of the following alternatives: “A”, “B”, “Aor B”, and “A and B”.

Various embodiments of the features of this disclosure are describedherein. However, it should be understood that such embodiments areprovided merely by way of example, and numerous variations, changes, andsubstitutions can occur to those skilled in the art without departingfrom the scope of this disclosure. It should also be understood thatvarious alternatives to the specific embodiments described herein arealso within the scope of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings illustrate certain embodiments of the featuresand advantages of this disclosure. These embodiments are not intended tolimit the scope of the appended claims in any manner. Like referencesymbols in the drawings indicate like elements.

FIG. 1 shows an exemplary spatial analysis workflow.

FIG. 2 is a schematic diagram showing an example of a barcoded captureprobe, as described herein.

FIG. 3 is a schematic illustrating a cleavable capture probe, whereinthe cleaved capture probe can enter into a non-permeabilized cell andbind to target analytes within the sample.

FIG. 4 is a schematic diagram of an exemplary multiplexedspatially-barcoded feature.

FIG. 5 is a schematic showing the exemplary arrangement of barcodedfeatures within an array.

FIG. 6 is a schematic diagram showing an exemplary workflow forRNA-templated ligation.

FIG. 7 is a schematic diagram showing an exemplary workflow forcapturing a ligation product on a substrate that includes captureprobes.

FIG. 8 is a schematic diagram showing an example of a first probe thatincludes a linker sequence and a second probe.

FIG. 9 is a schematic diagram showing an example of a second probe thatincludes a linker sequence and a first probe.

FIG. 10 is a schematic diagram showing an example of a first probe, asecond probe, and a spanning probe.

FIG. 11 is a schematic diagram showing an example of an RNA-templatedligation using a first probe oligonucleotide, a second probeoligonucleotide, and a third probe oligonucleotide.

FIGS. 12A-12E show various approaches for chemically-mediated nucleicacid ligation. FIG. 12A illustrates formation of a triazole bond. FIG.12B illustrates formation of a phosphorothioate bond. FIG. 12Cillustrates formation of an amide bond. FIG. 12D illustrates a formationof phosphoramidate bond. FIG. 12E illustrates a conjugation reaction.

FIG. 13 shows an exemplary RNA-templated ligation workflow.

FIG. 14 show enrichment PCR results for detection of various probes.R1=run 1; R2=run 2. (1) represents band for ligation product withcapture probe. (2), (3) and (4) represent non-ligation products.

FIG. 15 shows fraction of reads for various combination of conditionsand probes.

FIGS. 16A-16B show matrices of specific probe combinations.

FIGS. 17A-17C show gene expression patterns in mouse brain tissue usingtotal probe oligonucleotide counts (FIG. 17A), nonspecific probeoligonucleotide counts (FIG. 17B), and gene-specific probeoligonucleotide counts (FIG. 17C).

FIGS. 18A-18E show target genes with spatial expression in mouse braintissue.

FIGS. 19A-19F show gene expression patterns in mouse brain tissue usingtotal probe oligonucleotide counts (FIG. 19A) and gene-specific probeoligonucleotide (FIGS. 19B-19F).

FIG. 20A shows median unique molecular identifiers (UMIs) per cellversus mean reads per cell for different hybridization conditions.

FIG. 20B shows median genes per cell versus mean reads per cell fordifferent hybridization conditions.

FIG. 21A shows hematoxylin staining for a section of a triple positivebreast cancer tissue sample.

FIG. 21B shows median UMI per cell count versus mean read per cell forthe different conditions.

FIG. 21C shows t-SNE projection of spots in eight different clusters.

FIG. 21D shows the same tissue from FIG. 21A with various clusters (n=8clusters) expressed in distinct areas of the tissue.

FIG. 21E shows expression of estrogen receptor (ESR1).

FIG. 21F shows expression of estrogen receptor progesterone receptor(PGR).

FIG. 21G shows expression of ERBB2, also known as HER2.

FIG. 22A shows a hematoxylin stain for a section of Ovarian Cancersample.

FIG. 22B shows median UMI per cell versus mean read per cell for thedifferent conditions.

FIG. 22C shows the same tissue from FIG. 25A with various clusters (n=8clusters) expressed in distinct areas of the tissue.

FIG. 22D shows t-SNE projection of spots in eight different clusters.

FIG. 23 shows median UMIs per cell versus reads per cell for differentdecrosslinking conditions.

FIG. 24 shows median UMIs per cell versus reads per cell (top panel) andmedian genes per cell versus reads per cell (bottom panel) for differenttreatment conditions (RNase).

FIG. 25 shows median UMIs per cell versus reads per cell (top panel) andmedian genes per cell versus reads per cell (bottom panel) for differenttreatment conditions (time and temperature).

DETAILED DESCRIPTION

Targeted RNA capture is an attractive alternative to poly(A) mRNAcapture in order to interrogate spatial gene expression in FFPE tissue.Compared to poly(A) mRNA capture, targeted RNA capture is less affectedby RNA degradation associated with FFPE fixation than methods dependenton oligo-dT capture and reverse transcription of mRNA; allows forsensitive measurement of specific genes of interest that otherwise mightbe missed with a whole transcriptomic approach; and is scalable, withdemonstrated probes targeting a large fraction of the transcriptome.

Spatial analysis methodologies and compositions described herein canprovide a vast amount of analyte and/or expression data for a variety ofanalytes within a biological sample at high spatial resolution, whileretaining native spatial context. Spatial analysis methods andcompositions can include, e.g., the use of a capture probe including aspatial barcode (e.g., a nucleic acid sequence that provides informationas to the location or position of an analyte within a cell or a tissuesample (e.g., mammalian cell or a mammalian tissue sample) and a capturedomain that is capable of binding to an analyte (e.g., a protein and/ora nucleic acid) produced by and/or present in a cell. Spatial analysismethods and compositions can also include the use of a capture probehaving a capture domain that captures an intermediate agent for indirectdetection of an analyte. For example, the intermediate agent can includea nucleic acid sequence (e.g., a barcode) associated with theintermediate agent. Detection of the intermediate agent is thereforeindicative of the analyte in the cell or tissue sample.

Non-limiting aspects of spatial analysis methodologies and compositionsare described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022,10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810,9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent ApplicationPublication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641,2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709,2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322,2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875,2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee etal., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gaoet al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol.36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits UserGuide (e.g., Rev C, dated June 2020), and/or the Visium Spatial TissueOptimization Reagent Kits User Guide (e.g., Rev C, dated July 2020),both of which are available at the 10× Genomics Support Documentationwebsite, and can be used herein in any combination. Further non-limitingaspects of spatial analysis methodologies and compositions are describedherein.

Some general terminology that may be used in this disclosure can befound in WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. Typically, a “barcode” is a label, or identifier, thatconveys or is capable of conveying information (e.g., information aboutan analyte in a sample, a bead, and/or a capture probe). A barcode canbe part of an analyte, or independent of an analyte. A barcode can beattached to an analyte. A particular barcode can be unique relative toother barcodes. For the purpose of this disclosure, an “analyte” caninclude any biological substance, structure, moiety, or component to beanalyzed. The term “target” can similarly refer to an analyte ofinterest.

Analytes can be broadly classified into one of two groups: nucleic acidanalytes, and non-nucleic acid analytes. Examples of non-nucleic acidanalytes include, but are not limited to, lipids, carbohydrates,peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins,phosphoproteins, specific phosphorylated or acetylated variants ofproteins, amidation variants of proteins, hydroxylation variants ofproteins, methylation variants of proteins, ubiquitylation variants ofproteins, sulfation variants of proteins, viral proteins (e.g., viralcapsid, viral envelope, viral coat, viral accessory, viralglycoproteins, viral spike, etc.), extracellular and intracellularproteins, antibodies, and antigen binding fragments. In someembodiments, the analyte(s) can be localized to subcellular location(s),including, for example, organelles, e.g., mitochondria, Golgi apparatus,endoplasmic reticulum, chloroplasts, endocytic vesicles, exocyticvesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) canbe peptides or proteins, including without limitation antibodies andenzymes. Additional examples of analytes can be found in WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663. In someembodiments, an analyte can be detected indirectly, such as throughdetection of an intermediate agent, for example, a ligation product oran analyte capture agent (e.g., an oligonucleotide-conjugated antibody),such as those described herein.

A “biological sample” is typically obtained from the subject foranalysis using any of a variety of techniques including, but not limitedto, biopsy, surgery, and laser capture microscopy (LCM), and generallyincludes cells and/or other biological material from the subject. Insome embodiments, a biological sample can be a tissue section. In someembodiments, a biological sample can be a fixed and/or stainedbiological sample (e.g., a fixed and/or stained tissue section).Non-limiting examples of stains include histological stains (e.g.,hematoxylin and/or eosin) and immunological stains (e.g., fluorescentstains). In some embodiments, a biological sample (e.g., a fixed and/orstained biological sample) can be imaged. Biological samples are alsodescribed in WO 2020/176788 and/or U.S. Patent Application PublicationNo. 2020/0277663.

In some embodiments, a biological sample is permeabilized with one ormore permeabilization reagents. For example, permeabilization of abiological sample can facilitate analyte capture. Exemplarypermeabilization agents and conditions are described in WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663.

Array-based spatial analysis methods involve the transfer of one or moreanalytes from a biological sample to an array of features on asubstrate, where each feature is associated with a unique spatiallocation on the array. Subsequent analysis of the transferred analytesincludes determining the identity of the analytes and the spatiallocation of the analytes within the biological sample. The spatiallocation of an analyte within the biological sample is determined basedon the feature to which the analyte is bound (e.g., directly orindirectly) on the array, and the feature's relative spatial locationwithin the array.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain). In some embodiments,a capture probe can include a cleavage domain and/or a functional domain(e.g., a primer-binding site, such as for next-generation sequencing(NGS)). See, e.g., WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. Generation of capture probes can beachieved by any appropriate method, including those described in WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

In some embodiments, more than one analyte type (e.g., nucleic acids andproteins) from a biological sample can be detected (e.g., simultaneouslyor sequentially) using any appropriate multiplexing technique, such asthose described in WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663.

In some embodiments, detection of one or more analytes (e.g., proteinanalytes) can be performed using one or more analyte capture agents. Asused herein, an “analyte capture agent” refers to an agent thatinteracts with an analyte (e.g., an analyte in a biological sample) andwith a capture probe (e.g., a capture probe attached to a substrate or afeature) to identify the analyte. In some embodiments, the analytecapture agent includes: (i) an analyte binding moiety (e.g., that bindsto an analyte), for example, an antibody or antigen-binding fragmentthereof; (ii) analyte binding moiety barcode; and (iii) an analytecapture sequence. As used herein, the term “analyte binding moietybarcode” refers to a barcode that is associated with or otherwiseidentifies the analyte binding moiety. As used herein, the term “analytecapture sequence” refers to a region or moiety configured to hybridizeto, bind to, couple to, or otherwise interact with a capture domain of acapture probe. In some cases, an analyte binding moiety barcode (orportion thereof) may be able to be removed (e.g., cleaved) from theanalyte capture agent. Additional description of analyte capture agentscan be found in WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663.

There are at least two methods to associate a spatial barcode with oneor more neighboring cells, such that the spatial barcode identifies theone or more cells, and/or contents of the one or more cells, asassociated with a particular spatial location. One method is to promoteanalytes or analyte proxies (e.g., intermediate agents) out of a celland towards a spatially-barcoded array (e.g., includingspatially-barcoded capture probes). In some instances, thespatially-barcoded array populated with capture probes (as describedfurther herein) is contacted with a biological sample, and thebiological sample is permeabilized, allowing the analyte to migrate awayfrom the sample and toward the array. The analyte interacts with acapture probe on the spatially-barcoded array. Another method is tocleave spatially-barcoded capture probes from an array and promote thespatially-barcoded capture probes towards and/or into or onto thebiological sample. In some instances, the spatially-barcoded arraypopulated with capture probes (as described further herein) can becontacted with a sample. The spatially-barcoded capture probes arecleaved and then interact with cells within the provided biologicalsample. The interaction can be a covalent or non-covalent cell-surfaceinteraction. The interaction can be an intracellular interactionfacilitated by a delivery system or a cell penetration peptide. Once thespatially-barcoded capture probe is associated with a particular cell,the sample can be optionally removed for analysis. The sample can beoptionally dissociated before analysis. Once the tagged cell isassociated with the spatially-barcoded capture probe, the capture probescan be analyzed to obtain spatially-resolved information about thetagged cell.

In some instances, sample preparation may include placing the sample ona slide, fixing the sample, and/or staining the biological sample forimaging. The stained sample can be then imaged on the array using bothbrightfield (to image the sample hematoxylin and eosin stain) and/orfluorescence (to image features) modalities. Optionally, the sample canbe destained prior to permeabilization. In some embodiments, analytesare then released from the sample and capture probes forming thespatially-barcoded array hybridize or bind the released analytes. Thesample is then removed from the array and the capture probes cleavedfrom the array. The biological sample and array are then optionallyimaged a second time in one or both modalities while the analytes arereverse transcribed into cDNA, and an amplicon library is prepared andsequenced. Images are then spatially-overlaid in order to correlatespatially-identified biological sample information. When the sample andarray are not imaged a second time, a spot coordinate file is suppliedinstead. The spot coordinate file replaces the second imaging step.Further, amplicon library preparation can be performed with a unique PCRadapter and sequenced.

In some instances, disclosed is another exemplary workflow that utilizesa spatially-barcoded array on a substrate, where spatially-barcodedcapture probes are clustered at areas called features. Thespatially-barcoded capture probes can include a cleavage domain, one ormore functional domains, a spatial barcode, a unique molecularidentifier, and a capture domain. The spatially-barcoded capture probescan also include a 5′ end modification for reversible attachment to thesubstrate. The spatially-barcoded array is contacted with a biologicalsample, and the sample is permeabilized through application ofpermeabilization reagents. Permeabilization reagents may be administeredby placing the array/sample assembly within a bulk solution.Alternatively, permeabilization reagents may be administered to thesample via a diffusion-resistant medium and/or a physical barrier suchas a lid, wherein the sample is sandwiched between thediffusion-resistant medium and/or barrier and the array-containingsubstrate. The analytes are migrated toward the spatially-barcodedcapture array using any number of techniques disclosed herein. Forexample, analyte migration can occur using a diffusion-resistant mediumlid and passive migration. As another example, analyte migration can beactive migration, using an electrophoretic transfer system, for example.Once the analytes are in close proximity to the spatially-barcodedcapture probes, the capture probes can hybridize or otherwise bind atarget analyte. The biological sample can be optionally removed from thearray.

The capture probes can be optionally cleaved from the array, and thecaptured analytes can be spatially-barcoded by performing a reversetranscriptase first strand cDNA reaction. A first strand cDNA reactioncan be optionally performed using template switching oligonucleotides.For example, a template switching oligonucleotide can hybridize to apoly(C) tail added to a 3′ end of the cDNA by a reverse transcriptaseenzyme in a template independent manner. The original mRNA template andtemplate switching oligonucleotide can then be denatured from the cDNAand the spatially-barcoded capture probe can then hybridize with thecDNA and a complement of the cDNA can be generated. The first strandcDNA can then be purified and collected for downstream amplificationsteps. The first strand cDNA can be amplified using PCR, where theforward and reverse primers flank the spatial barcode and analyteregions of interest, generating a library associated with a particularspatial barcode. In some embodiments, the library preparation can bequantitated and/or quality controlled to verify the success of thelibrary preparation steps. In some embodiments, the cDNA comprises asequencing by synthesis (SBS) primer sequence. The library amplicons aresequenced and analyzed to decode spatial information.

In some cases, capture probes may be configured to prime, replicate, andconsequently yield optionally barcoded extension products from atemplate (e.g., a DNA or RNA template, such as an analyte or anintermediate agent (e.g., a ligation product or an analyte captureagent), or a portion thereof), or derivatives thereof (see, e.g., WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663regarding extended capture probes). In some cases, capture probes may beconfigured to form ligation products with a template (e.g., a DNA or RNAtemplate, such as an analyte or an intermediate agent, or portionthereof), thereby creating ligations products that serve as proxies fora template.

A “capture probe” refers to any molecule capable of capturing (directlyor indirectly) and/or labelling an analyte (e.g., an analyte ofinterest) in a biological sample. In some embodiments, the capture probeis a nucleic acid or a polypeptide. In some embodiments, the captureprobe includes a barcode (e.g., a spatial barcode and/or a uniquemolecular identifier (UMI)) and a capture domain. In some instances, thecapture probe can include functional sequences that are useful forsubsequent processing. In some instances, a capture probe can bereversibly attached to a substrate via a linker. The capture probe caninclude one or more functional sequences, which can include a sequencerspecific flow cell attachment sequence, e.g., a P5 or P7 sequence, aswell as functional sequence, which can include sequencing primersequences, e.g., a R1 primer binding site, a R2 primer binding site. Insome embodiments, sequence is a P7 sequence and sequence is a R2 primerbinding site. A capture probe can additionally include a spatial barcodeand/or unique molecular identifier and a capture domain. The differentsequences of the capture probe need not be in the sequential manner asdepicted in this example, however the capture domain should be placed ina location on the barcode wherein analyte capture and extension of thecapture domain to create a copy of the analyte can occur.

FIG. 2 is a schematic diagram showing an example of a capture probe, asdescribed herein. As shown, the capture probe 202 is optionally coupledto a feature 201 by a cleavage domain 203, such as a disulfide linker.The capture probe can include functional sequences that are useful forsubsequent processing, such as functional sequence 204, which caninclude a sequencer specific flow cell attachment sequence, e.g., a P5or P7 sequence, as well as functional sequence 205, which can includesequencing primer sequences, e.g., a R1 primer binding site. In someembodiments, sequence 204 is a P7 sequence and sequence 205 is a R1primer binding site. A spatial barcode 206 can be included within thecapture probe for use in barcoding the target analyte. The functionalsequences can generally be selected for compatibility with any of avariety of different sequencing systems, e.g., Ion Torrent Proton orPGM, Illumina sequencing instruments, PacBio, Oxford Nanopore, etc., andthe requirements thereof. In some embodiments, functional sequences canbe selected for compatibility with non-commercialized sequencingsystems. Examples of such sequencing systems and techniques, for whichsuitable functional sequences can be used, include (but are not limitedto) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBioSMRT sequencing, and Oxford Nanopore sequencing. Further, in someembodiments, functional sequences can be selected for compatibility withother sequencing systems.

In some embodiments, the spatial barcode 206, functional sequences 204(e.g., flow cell attachment sequence) and 205 (e.g., sequencing primersequences) can be common to all of the probes attached to a givenfeature. The spatial barcode can also include a capture domain 207 tofacilitate capture of a target analyte.

In some cases, capture probes are introduced into the cell using acell-penetrating peptide. FIG. 3 is a schematic illustrating a cleavablecapture probe that includes a cell-penetrating peptide, wherein thecleaved capture probe can enter into a non-permeabilized cell and bindto analytes within the sample. The capture probe 301 contains a cleavagedomain 302, a cell penetrating peptide 303, a reporter molecule 304, anda disulfide bond (—S—S—). 305 represents all other parts of a captureprobe, for example a spatial barcode and a capture domain.

In some instances, the disclosure provides multiplexedspatially-barcoded features. FIG. 4 is a schematic diagram of anexemplary multiplexed spatially-barcoded feature. In FIG. 4, the feature401 (e.g., a bead, a location on a slide or other substrate, a well on aslide or other substrate, a partition on a slide or other substrate,etc.) can be coupled to spatially-barcoded capture probes, wherein thespatially-barcoded probes of a particular feature can possess the samespatial barcode, but have different capture domains designed toassociate the spatial barcode of the feature with more than one targetanalyte. For example, a feature may be coupled to four different typesof spatially-barcoded capture probes, each type of spatially-barcodedcapture probe possessing the spatial barcode 402. One type of captureprobe associated with the feature includes the spatial barcode 402 incombination with a poly(T) capture domain 403, designed to capture mRNAtarget analytes. A second type of capture probe associated with thefeature includes the spatial barcode 402 in combination with a randomN-mer capture domain 404 for gDNA analysis. A third type of captureprobe associated with the feature includes the spatial barcode 402 incombination with a capture domain complementary to the analyte captureagent of interest 405. A fourth type of capture probe associated withthe feature includes the spatial barcode 402 in combination with acapture probe that can specifically bind a nucleic acid molecule 406that can function in a CRISPR assay (e.g., CRISPR/Cas9). While only fourdifferent capture probe-barcoded constructs are shown in FIG. 4,capture-probe barcoded constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct. For example, the schemes shown in FIG. 4 can also beused for concurrent analysis of other analytes disclosed herein,including, but not limited to: (a) mRNA, a lineage tracing construct,cell surface or intracellular proteins and metabolites, and gDNA; (b)mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq)cell surface or intracellular proteins and metabolites, and aperturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc fingernuclease, and/or antisense oligonucleotide as described herein); (c)mRNA, cell surface or intracellular proteins and/or metabolites, abarcoded labelling agent (e.g., the MHC multimers described herein), anda V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). Insome embodiments, a perturbation agent can be a small molecule, anantibody, a drug, an aptamer, a miRNA, a physical environmental (e.g.,temperature change), or any other known perturbation agents.

Additional features of capture probes are described in WO 2020/176788and/or U.S. Patent Application Publication No. 2020/0277663, each ofwhich is incorporated by reference in its entirety. Generation ofcapture probes can be achieved by any appropriate method, includingthose described in WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663, each of which is incorporated by referencein its entirety.

As used herein, an “extended capture probe” refers to a capture probehaving additional nucleotides added to the terminus (e.g., 3′ or 5′ end)of the capture probe thereby extending the overall length of the captureprobe. For example, an “extended 3′ end” indicates additionalnucleotides were added to the most 3′ nucleotide of the capture probe toextend the length of the capture probe, for example, by polymerizationreactions used to extend nucleic acid molecules including templatedpolymerization catalyzed by a polymerase (e.g., a DNA polymerase or areverse transcriptase). In some embodiments, extending the capture probeincludes adding to a 3′ end of a capture probe a nucleic acid sequencethat is complementary to a nucleic acid sequence of an analyte orintermediate agent specifically bound to the capture domain of thecapture probe. In some embodiments, the capture probe is extended usingreverse transcription. In some embodiments, the capture probe isextended using one or more DNA polymerases. The extended capture probesinclude the sequence of the capture probe and the sequence of thespatial barcode of the capture probe. In some embodiments, extendedcapture probes are amplified (e.g., in bulk solution or on the array) toyield quantities that are sufficient for downstream analysis, e.g., viaDNA sequencing. In some embodiments, extended capture probes (e.g., DNAmolecules) act as templates for an amplification reaction (e.g., apolymerase chain reaction).

Additional variants of spatial analysis methods, including in someembodiments, an imaging step, are described in WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663. Analysis ofcaptured analytes (and/or intermediate agents or portions thereof), forexample, including sample removal, extension of capture probes,sequencing (e.g., of a cleaved extended capture probe and/or a cDNAmolecule complementary to an extended capture probe), sequencing on thearray (e.g., using, for example, in situ hybridization or in situligation approaches), temporal analysis, and/or proximity capture, isdescribed in WO 2020/176788 and/or U.S. Patent Application PublicationNo. 2020/0277663. Some quality control measures are described in WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.

Spatial information can provide information of biological and/or medicalimportance. For example, the methods and compositions described hereincan allow for: identification of one or more biomarkers (e.g.,diagnostic, prognostic, and/or for determination of efficacy of atreatment) of a disease or disorder; identification of a candidate drugtarget for treatment of a disease or disorder; identification (e.g.,diagnosis) of a subject as having a disease or disorder; identificationof stage and/or prognosis of a disease or disorder in a subject;identification of a subject as having an increased likelihood ofdeveloping a disease or disorder; monitoring of progression of a diseaseor disorder in a subject; determination of efficacy of a treatment of adisease or disorder in a subject; identification of a patientsubpopulation for which a treatment is effective for a disease ordisorder; modification of a treatment of a subject with a disease ordisorder; selection of a subject for participation in a clinical trial;and/or selection of a treatment for a subject with a disease ordisorder.

Spatial information can provide information of biological importance.For example, the methods and compositions described herein can allowfor: identification of transcriptome and/or proteome expression profiles(e.g., in healthy and/or diseased tissue); identification of multipleanalyte types in close proximity (e.g., nearest neighbor analysis);determination of up- and/or down-regulated genes and/or proteins indiseased tissue; characterization of tumor microenvironments;characterization of tumor immune responses; characterization of cellstypes and their co-localization in tissue; and identification of geneticvariants within tissues (e.g., based on gene and/or protein expressionprofiles associated with specific disease or disorder biomarkers).

Typically, for spatial array-based methods, a substrate functions as asupport for direct or indirect attachment of capture probes to featuresof the array. A “feature” is an entity that acts as a support orrepository for various molecular entities used in spatial analysis. Insome embodiments, some or all of the features in an array arefunctionalized for analyte capture. Exemplary substrates are describedin WO 2020/176788 and/or U.S. Patent Application Publication No.2020/0277663. Exemplary features and geometric attributes of an arraycan be found in WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663.

FIG. 5 depicts an exemplary arrangement of barcoded features within anarray. From left to right, FIG. 5 shows (left) a slide including sixspatially-barcoded arrays, (center) an enlarged schematic of one of thesix spatially-barcoded arrays, showing a grid of barcoded features inrelation to a biological sample, and (right) an enlarged schematic ofone section of an array, showing the specific identification of multiplefeatures within the array (labelled as ID578, ID579, ID560, etc.).

Generally, analytes and/or intermediate agents (or portions thereof) canbe captured when contacting a biological sample with a substrateincluding capture probes (e.g., a substrate with capture probesembedded, spotted, printed, fabricated on the substrate, or a substratewith features (e.g., beads, wells, areas on a substrate) comprisingcapture probes). As used herein, “contact,” “contacted,” and/or“contacting,” a biological sample with a substrate refers to any contact(e.g., direct or indirect) such that capture probes can interact (e.g.,bind covalently or non-covalently (e.g., hybridize)) with analytes fromthe biological sample. Capture can be achieved actively (e.g., usingelectrophoresis) or passively (e.g., using diffusion). Analyte captureis further described in WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663.

In some cases, spatial analysis can be performed by attaching and/orintroducing a molecule (e.g., a peptide, a lipid, or a nucleic acidmolecule) having a barcode (e.g., a spatial barcode) to a biologicalsample (e.g., to a cell in a biological sample). In some embodiments, aplurality of molecules (e.g., a plurality of nucleic acid molecules)having a plurality of barcodes (e.g., a plurality of spatial barcodes)are introduced to a biological sample (e.g., to a plurality of cells ina biological sample) for use in spatial analysis. In some embodiments,after attaching and/or introducing a molecule having a barcode to abiological sample, the biological sample can be physically separated(e.g., dissociated) into single cells or cell groups for analysis. Somesuch methods of spatial analysis are described in WO 2020/176788 and/orU.S. Patent Application Publication No. 2020/0277663.

In some cases, spatial analysis can be performed by detecting multipleoligonucleotides that hybridize to an analyte. In some instances, forexample, spatial analysis can be performed using RNA-templated ligation(RTL). Methods of RTL have been described previously. See, e.g., Credleet al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTLincludes hybridization of two oligonucleotides to adjacent sequences onan analyte (e.g., an RNA molecule, such as an mRNA molecule). In someinstances, the oligonucleotides are DNA molecules. In some instances,one of the oligonucleotides includes at least two ribonucleic acid basesat the 3′ end and/or the other oligonucleotide includes a phosphorylatednucleotide at the 5′ end. In some instances, one of the twooligonucleotides includes a capture domain (e.g., a poly(A) sequence, anon-homopolymeric sequence). After hybridization to the analyte, aligase (e.g., SplintR ligase) ligates the two oligonucleotides together,creating a ligation product. In some instances, the two oligonucleotideshybridize to sequences that are not adjacent to one another. Forexample, hybridization of the two oligonucleotides creates a gap betweenthe hybridized oligonucleotides. In some instances, a polymerase (e.g.,a DNA polymerase) can extend one of the oligonucleotides prior toligation. After ligation, the ligation product is released from theanalyte. In some instances, the ligation product is released using anendonuclease (e.g., RNAse H). The released ligation product can then becaptured by capture probes (e.g., instead of direct capture of ananalyte) on an array, optionally amplified, and sequenced, thusdetermining the location and optionally the abundance of the analyte inthe biological sample.

During analysis of spatial information, sequence information for aspatial barcode associated with an analyte is obtained, and the sequenceinformation can be used to provide information about the spatialdistribution of the analyte in the biological sample. Various methodscan be used to obtain the spatial information. In some embodiments,specific capture probes and the analytes they capture are associatedwith specific locations in an array of features on a substrate. Forexample, specific spatial barcodes can be associated with specific arraylocations prior to array fabrication, and the sequences of the spatialbarcodes can be stored (e.g., in a database) along with specific arraylocation information, so that each spatial barcode uniquely maps to aparticular array location.

Alternatively, specific spatial barcodes can be deposited atpredetermined locations in an array of features during fabrication suchthat at each location, only one type of spatial barcode is present sothat spatial barcodes are uniquely associated with a single feature ofthe array. Where necessary, the arrays can be decoded using any of themethods described herein so that spatial barcodes are uniquelyassociated with array feature locations, and this mapping can be storedas described above.

When sequence information is obtained for capture probes and/or analytesduring analysis of spatial information, the locations of the captureprobes and/or analytes can be determined by referring to the storedinformation that uniquely associates each spatial barcode with an arrayfeature location. In this manner, specific capture probes and capturedanalytes are associated with specific locations in the array offeatures. Each array feature location represents a position relative toa coordinate reference point (e.g., an array location, a fiducialmarker) for the array. Accordingly, each feature location has an“address” or location in the coordinate space of the array.

Some exemplary spatial analysis workflows are described in WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.See, for example, the Exemplary embodiment starting with “In somenon-limiting examples of the workflows described herein, the sample canbe immersed . . . ” of WO 2020/176788 and/or U.S. Patent ApplicationPublication No. 2020/0277663. See also, e.g., the Visium Spatial GeneExpression Reagent Kits User Guide (e.g., Rev C, dated June 2020),and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide(e.g., Rev C, dated July 2020).

In some embodiments, spatial analysis can be performed using dedicatedhardware and/or software, such as any of the systems described in WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663,or any of one or more of the devices or methods described in WO2020/123320.

Suitable systems for performing spatial analysis can include componentssuch as a chamber (e.g., a flow cell or sealable, fluid-tight chamber)for containing a biological sample. The biological sample can be mountedfor example, in a biological sample holder. One or more fluid chamberscan be connected to the chamber and/or the sample holder via fluidconduits, and fluids can be delivered into the chamber and/or sampleholder via fluidic pumps, vacuum sources, or other devices coupled tothe fluid conduits that create a pressure gradient to drive fluid flow.One or more valves can also be connected to fluid conduits to regulatethe flow of reagents from reservoirs to the chamber and/or sampleholder.

The systems can optionally include a control unit that includes one ormore electronic processors, an input interface, an output interface(such as a display), and a storage unit (e.g., a solid state storagemedium such as, but not limited to, a magnetic, optical, or other solidstate, persistent, writeable and/or re-writeable storage medium). Thecontrol unit can optionally be connected to one or more remote devicesvia a network. The control unit (and components thereof) can generallyperform any of the steps and functions described herein. Where thesystem is connected to a remote device, the remote device (or devices)can perform any of the steps or features described herein. The systemscan optionally include one or more detectors (e.g., CCD, CMOS) used tocapture images. The systems can also optionally include one or morelight sources (e.g., LED-based, diode-based, lasers) for illuminating asample, a substrate with features, analytes from a biological samplecaptured on a substrate, and various control and calibration media.

The systems can optionally include software instructions encoded and/orimplemented in one or more of tangible storage media and hardwarecomponents such as application specific integrated circuits. Thesoftware instructions, when executed by a control unit (and inparticular, an electronic processor) or an integrated circuit, can causethe control unit, integrated circuit, or other component executing thesoftware instructions to perform any of the method steps or functionsdescribed herein.

In some cases, the systems described herein can detect (e.g., registeran image) the biological sample on the array. Exemplary methods todetect the biological sample on an array are described in PCTApplication No. 2020/061064 and/or U.S. patent application Ser. No.16/951,854.

Prior to transferring analytes from the biological sample to the arrayof features on the substrate, the biological sample can be aligned withthe array. Alignment of a biological sample and an array of featuresincluding capture probes can facilitate spatial analysis, which can beused to detect differences in analyte presence and/or level withindifferent positions in the biological sample, for example, to generate athree-dimensional map of the analyte presence and/or level. Exemplarymethods to generate a two- and/or three-dimensional map of the analytepresence and/or level are described in PCT Application No. 2020/053655and spatial analysis methods are generally described in WO 2020/061108and/or U.S. patent application Ser. No. 16/951,864.

In some cases, a map of analyte presence and/or level can be aligned toan image of a biological sample using one or more fiducial markers,e.g., objects placed in the field of view of an imaging system whichappear in the image produced, as described in WO 2020/123320, PCTApplication No. 2020/061066, and/or U.S. patent application Ser. No.16/951,843. Fiducial markers can be used as a point of reference ormeasurement scale for alignment (e.g., to align a sample and an array,to align two substrates, to determine a location of a sample or array ona substrate relative to a fiducial marker) and/or for quantitativemeasurements of sizes and/or distances.

I. RNA Capture Using RNA-Templated Ligation

(A) General Background

Although techniques such as whole genome sequencing and whole exomesequencing are available, these techniques have drawbacks in that theyprovide a lot of information and increase costs for an experiment. Insituations where one prefers to examine a more limited number ofanalytes, methods herein are provided for targeted RNA capture.Capturing a derivative of an analyte (e.g., a ligation product) providesenhanced specificity with respect to detection of an analyte. This isbecause at least two probes specific for a target are required tohybridize to the target in order to facilitate ligation and ultimatecapture of the nucleic acid.

Referring to FIG. 1, in an exemplary embodiment of the disclosure,provided are methods for identifying a location of an analyte in abiological sample. In some instances, the methods include 101 contactinga biological sample with array of spatially-barcoded capture probes. Insome instances, the array is on a substrate and the array includes aplurality of capture probes, wherein a capture probe of the pluralityincludes: (i) a spatial barcode and (ii) a capture domain. After placingthe biological sample on the array, the biological sample 102 iscontacted with a first probe and a second probe, wherein the first probeand the second probe each include one or more sequences that aresubstantially complementary to sequences of the analyte, and wherein thesecond probe includes a capture probe capture domain; the first probeand the second probe 103 hybridize to complementary sequences in theanalyte. After hybridization a ligation product comprising the firstprobe and the second probe 104 is generated, and the ligation product isreleased from the analyte. The liberated ligation product is then freed105 to hybridize to the capture domain of a probe on the array. Aftercapture, (i) all or a part of the sequence of the ligation productspecifically bound to the capture domain, or a complement thereof, and(ii) all or a part of the sequence of the spatial barcode, or acomplement thereof 106 can be determined, and then one can use thedetermined sequence of (i) and (ii) 107 to identify the location of theanalyte in the biological sample.

Referring to FIG. 13, in another non-limiting example, a biologicalsample is deparaffinized, stained, and imaged 1301. After destaining anddecrosslinking 1302, probes are added to the sample and hybridize to ananalyte 1303. In some instances, the probes are DNA probes. In someinstances, the probes are diribo-containing probes. Probes are ligated1304 and then released using an endonuclease such as RNAse H 1305.Ligated probes are captured on an array by a capture probe 1306,extended using a polymerase 1307 and denatured 1308. After qualitycontrol cleanup 1309, the abundance and location of an analyte isdetermined.

A non-limiting example of the methods disclosed herein is depicted inFIG. 6. After a biological sample is contacted with a substrateincluding a plurality of capture probes and contacted with (a) a firstprobe 601 having a target-hybridization sequence 603 and a primersequence 602 and (b) a second probe 604 having a target-hybridizationsequence 605 and a capture domain (e.g., a poly-A sequence) 606, thefirst probe 601 and a second probe 604 hybridize 610 to an analyte 607.A ligase 621 ligates 620 the first probe to the second probe therebygenerating a ligation product 622. The ligation product is released 630from the analyte 631 by digesting the analyte using an endoribonuclease632. The sample is permeabilized 640 and the ligation product 641 isable to hybridize to a capture probe on the substrate.

Also provided herein are methods for identifying a location of ananalyte in a biological sample that includes a second probe including apre-adenylated phosphate group at its 5′ end, which enables the ligatingto use a ligase that does not require adenosine triphosphate for ligaseactivity.

Also provided herein are methods for identifying a location of ananalyte in a biological sample that includes one or more spanningprobes, in addition to the first and second probes. Using a spanningprobe enables greater flexibility in designing RTL probes, primarily byincreasing the sequences within the analyte that can be used as optionaltarget sequences.

Also provided herein are methods for identifying a location of ananalyte in a biological sample that includes optimized hybridizing,washing, and releasing steps.

In some embodiments, as shown in FIG. 7, the ligation product 701includes a capture probe capture domain 702, which can bind to a captureprobe 703 (e.g., a capture probe immobilized, directly or indirectly, ona substrate 704). In some embodiments, methods provided herein includecontacting 705 a biological sample with a substrate 704, wherein thecapture probe 703 is affixed to the substrate (e.g., immobilized to thesubstrate, directly or indirectly). In some embodiments, the captureprobe capture domain 702 of the ligated product specifically binds tothe capture domain 706. The capture probe can also include a uniquemolecular identifier (UMI) 707, a spatial barcode 708, a functionalsequence 709, and a cleavage domain 710.

In some embodiments, methods provided herein include permeabilization ofthe biological sample such that the capture probe can more easily bindto the captured ligated probe (i.e., compared to no permeabilization).In some embodiments, reverse transcription (RT) reagents can be added topermeabilized biological samples. Incubation with the RT reagents canextend the capture probes 711 to produce spatially-barcoded full-lengthcDNA 712 and 713 from the captured analytes (e.g., polyadenylated mRNA).Second strand reagents (e.g., second strand primers, enzymes) can beadded to the biological sample on the slide to initiate second strandsynthesis.

In some embodiments, cDNA can be denatured 714 from the capture probetemplate and transferred (e.g., to a clean tube) for amplification,and/or library construction. The spatially-barcoded, full-length cDNAcan be amplified 715 via PCR prior to library construction. The cDNA canthen be enzymatically fragmented and size-selected in order to optimizethe cDNA amplicon size. P5 716, i5 717, i7 718, and P7 719, and can beused as sample indexes, and TruSeq Read 2 can be added via End Repair,A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then besequenced using paired-end sequencing using TruSeq Read 1 and TruSeqRead 2 as sequencing primer sites.

(b) Probes for RNA-Templated Ligation

The methods provided herein utilize probe pairs (or sets; the terms areinterchangeable). In some instances, the probe pairs are designed sothat each probe hybridizes to a sequence in an analyte that is specificto the analyte (e.g., compared to the entire genome). That is, in someinstances, a single probe pair can be specific to a single analyte.

In other embodiments, probes can be designed so that one of the probesof a pair is a probe that hybridizes to a specific sequence. Then, theother probe can be designed to detect a mutation of interest.Accordingly, in some instances, multiple second probes can be designedand can vary so that each binds to a specific sequence. For example, onesecond probe can be designed to hybridize to a wild-type sequence, andanother second probe can be designed to detect a mutated sequence. Thus,in some instances, a probe set can include one first probe and twosecond probes (or vice versa).

On the other hand, in some instances, probes can be designed so thatthey cover conserved regions of an analyte. Thus, in some instances, aprobe (or probe pair can hybridize to similar analytes in a biologicalsample (e.g., to detect conserved or similar analytes) or in differentbiological samples (e.g., across different species).

In some embodiments, probe sets cover all or nearly all of a genome(e.g., human genome). In instances where probe sets are designed tocover an entire genome (e.g., the human genome), the methods disclosedherein can detect analytes in an unbiased manner. In some instances, oneprobe oligonucleotide pair is designed to cover one analyte (e.g.,transcript). In some instances, more than one probe oligonucleotide pair(e.g., a probe pair comprising a first probe and a second probe) isdesigned to cover one analyte (e.g., transcript). For example, at leasttwo, three, four, five, six, seven, eight, nine, ten, or more probe setscan be used to hybridize to a single analyte. Factors to consider whendesigning probes is presence of variants (e.g., SNPs, mutations) ormultiple isoforms expressed by a single gene. In some instances, theprobe oligonucleotide pair does not hybridize to the entire analyte(e.g., a transcript), but instead the probe oligonucleotide pairhybridizes to a portion of the entire analyte (e.g., transcript).

In some instances, about 5000, 10,000, 15,000, 20,000, or more probeoligonucleotides pair (e.g., a probe pair comprising a first probe and asecond probe) are used in the methods described herein. In someinstances, about 20,000 probe oligonucleotides pair are used in themethods described herein

In some instances, RNA capture is targeted RNA capture. Targeted RNAcapture using the methods disclosed herein allows for examination of asubset of RNA analytes from the entire transcriptome. In someembodiments, the subset of analytes includes an individual target RNA.In some embodiments, the subset of analytes includes two or moretargeted RNAs. In some embodiments, the subset of analytes includes oneor more mRNAs transcribed by one or more targeted genes. In someembodiments, the subset of analytes includes one or more mRNA splicevariants of one or more targeted genes. In some embodiments, the subsetof analytes includes non-polyadenylated RNAs in a biological sample. Insome embodiments, the subset of analytes includes detection of mRNAshaving one or more single nucleotide polymorphisms (SNPs) in abiological sample.

In some embodiments, the subset of analytes includes mRNAs that mediateexpression of a set of genes of interest. In some embodiments, thesubset of analytes includes mRNAs that share identical or substantiallysimilar sequences, which mRNAs are translated into polypeptides havingsimilar functional groups or protein domains. In some embodiments, thesubset of analytes includes mRNAs that do not share identical orsubstantially similar sequences, which mRNAs are translated intoproteins that do not share similar functional groups or protein domains.In some embodiments, the subset of analytes includes mRNAs that aretranslated into proteins that function in the same or similar biologicalpathways. In some embodiments, the biological pathways are associatedwith a pathologic disease. For example, targeted RNA capture can detectgenes that are overexpressed or underexpressed in cancer.

In some embodiments, the subset of analytes includes 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, about 100, about 110,about 120, about 130, about 140, about 150, about 160, about 170, about180, about 190, about 200, about 225, about 250, about 275, about 300,about 325, about 350, about 375, about 400, about 425, about 450, about475, about 500, about 600, about 700, about 800, about 900, or about1000 analytes.

In some instances, the methods disclosed herein can detect the abundanceand location of at least 5,000, 10,000, 15,000, 20,000, or moredifferent analytes.

In some embodiments, the subset of analytes detected by targeted RNAcapture methods provided herein includes a large proportion of thetranscriptome of one or more cells. For example, the subset of analytesdetected by targeted RNA capture methods provided herein can include atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more of the mRNAs present in thetranscriptome of one or more cells.

In some instances, the probes are DNA probes. In some instances, theprobes are diribo-containing probes.

Additional embodiments of probe(s) and probe set(s) are describedherein.

(i) First Probe

In some embodiments, the methods described herein include a first probe.As used herein, a “first probe” can refer to a probe that hybridizes toall or a portion of an analyte and can be ligated to one or moreadditional probes (e.g., a second probe or a spanning probe). In someembodiments, “first probe” can be used interchangeably with “first probeoligonucleotide.”

In some embodiments, the first probe includes ribonucleotides,deoxyribonucleotides, and/or synthetic nucleotides that are capable ofparticipating in Watson-Crick type or analogous base pair interactions.In some embodiments, the first probe includes deoxyribonucleotides. Insome embodiments, the first probe includes deoxyribonucleotides andribonucleotides. In some embodiments, the first probe includes adeoxyribonucleic acid that hybridizes to an analyte, and includes aportion of the oligonucleotide that is not a deoxyribonucleic acid. Forexample, in some embodiments, the portion of the first oligonucleotidethat is not a deoxyribonucleic acid is a ribonucleic acid or any othernon-deoxyribonucleic acid nucleic acid as described herein. In someembodiments where the first probe includes deoxyribonucleotides,hybridization of the first probe to the mRNA molecule results in aDNA:RNA hybrid. In some embodiments, the first probe includes onlydeoxyribonucleotides and upon hybridization of the first probe to themRNA molecule results in a DNA:RNA hybrid.

In some embodiments, the method includes a first probe that includes oneor more sequences that are substantially complementary to one or moresequences of an analyte. In some embodiments, a first probe includes asequence that is substantially complementary to a first target sequencein the analyte. In some embodiments, the sequence of the first probethat is substantially complementary to the first target sequence in theanalyte is at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% complementary to the first target sequence in the analyte.

In some embodiments, a first probe includes a sequence that is about 10nucleotides to about 100 nucleotides (e.g., a sequence of about 10nucleotides to about 90 nucleotides, about 10 nucleotides to about 80nucleotides, about 10 nucleotides to about 70 nucleotides, about 10nucleotides to about 60 nucleotides, about 10 nucleotides to about 50nucleotides, about 10 nucleotides to about 40 nucleotides, about 10nucleotides to about 30 nucleotides, about 10 nucleotides to about 20nucleotides, about 20 nucleotides to about 100 nucleotides, about 20nucleotides to about 90 nucleotides, about 20 nucleotides to about 80nucleotides, about 20 nucleotides to about 70 nucleotides, about 20nucleotides to about 60 nucleotides, about 20 nucleotides to about 50nucleotides, about 20 nucleotides to about 40 nucleotides, about 20nucleotides to about 30 nucleotides, about 30 nucleotides to about 100nucleotides, about 30 nucleotides to about 90 nucleotides, about 30nucleotides to about 80 nucleotides, about 30 nucleotides to about 70nucleotides, about 30 nucleotides to about 60 nucleotides, about 30nucleotides to about 50 nucleotides, about 30 nucleotides to about 40nucleotides, about 40 nucleotides to about 100 nucleotides, about 40nucleotides to about 90 nucleotides, about 40 nucleotides to about 80nucleotides, about 40 nucleotides to about 70 nucleotides, about 40nucleotides to about 60 nucleotides, about 40 nucleotides to about 50nucleotides, about 50 nucleotides to about 100 nucleotides, about 50nucleotides to about 90 nucleotides, about 50 nucleotides to about 80nucleotides, about 50 nucleotides to about 70 nucleotides, about 50nucleotides to about 60 nucleotides, about 60 nucleotides to about 100nucleotides, about 60 nucleotides to about 90 nucleotides, about 60nucleotides to about 80 nucleotides, about 60 nucleotides to about 70nucleotides, about 70 nucleotides to about 100 nucleotides, about 70nucleotides to about 90 nucleotides, about 70 nucleotides to about 80nucleotides, about 80 nucleotides to about 100 nucleotides, about 80nucleotides to about 90 nucleotides, or about 90 nucleotides to about100 nucleotides).

In some embodiments, a sequence of the first probe that is substantiallycomplementary to a sequence in the analyte includes a sequence that isabout 5 nucleotides to about 50 nucleotides (e.g., about 5 nucleotidesto about 45 nucleotides, about 5 nucleotides to about 40 nucleotides,about 5 nucleotides to about 35 nucleotides, about 5 nucleotides toabout 30 nucleotides, about 5 nucleotides to about 25 nucleotides, about5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 15nucleotides, about 5 nucleotides to about 10 nucleotides, about 10nucleotides to about 50 nucleotides, about 10 nucleotides to about 45nucleotides, about 10 nucleotides to about 40 nucleotides, about 10nucleotides to about 35 nucleotides, about 10 nucleotides to about 30nucleotides, about 10 nucleotides to about 25 nucleotides, about 10nucleotides to about 20 nucleotides, about 10 nucleotides to about 15nucleotides, about 15 nucleotides to about 50 nucleotides, about 15nucleotides to about 45 nucleotides, about 15 nucleotides to about 40nucleotides, about 15 nucleotides to about 35 nucleotides, about 15nucleotides to about 30 nucleotides, about 15 nucleotides to about 25nucleotides, about 15 nucleotides to about 20 nucleotides, about 20nucleotides to about 50 nucleotides, about 20 nucleotides to about 45nucleotides, about 20 nucleotides to about 40 nucleotides, about 20nucleotides to about 35 nucleotides, about 20 nucleotides to about 30nucleotides, about 20 nucleotides to about 25 nucleotides, about 25nucleotides to about 50 nucleotides, about 25 nucleotides to about 45nucleotides, about 25 nucleotides to about 40 nucleotides, about 25nucleotides to about 35 nucleotides, about 25 nucleotides to about 30nucleotides, about 30 nucleotides to about 50 nucleotides, about 30nucleotides to about 45 nucleotides, about 30 nucleotides to about 40nucleotides, about 30 nucleotides to about 35 nucleotides, about 35nucleotides to about 50 nucleotides, about 35 nucleotides to about 45nucleotides, about 35 nucleotides to about 40 nucleotides, about 40nucleotides to about 50 nucleotides, about 40 nucleotides to about 45nucleotides, or about 45 nucleotides to about 50 nucleotides).

In some embodiments, a first probe includes a functional sequence. Insome embodiments, a functional sequence includes a primer sequence.

In some embodiments, a first probe includes at least two ribonucleicacid bases at the 3′ end. In such cases, a second probe oligonucleotidecomprises a phosphorylated nucleotide at the 5′ end. In someembodiments, a first probe includes at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,or at least ten ribonucleic acid bases at the 3′ end.

As shown in FIG. 6, a non-limiting example of a first probe 601, whichcan be referred to as an LHS probe, includes a functional sequence 602,a sequence 603 that is substantially complementary to a first targetsequence in the analyte 607, and two ribonucleic acid bases at the 3′end.

In some embodiments, a first probe includes an auxiliary sequence thatdoes not hybridize to an analyte. In some embodiments, the auxiliarysequence can be used to hybridize to additional probes.

(ii) Second Probe

In some embodiments, the methods described herein include a secondprobe. As used herein, a “second probe” can refer to a probe thathybridizes to all or a portion of an analyte and can be ligated to oneor more additional probes (e.g., a first probe or a spanning probe). Insome embodiments, “second probe” can be used interchangeably with“second probe oligonucleotide.” One of skill in the art will appreciatethat the order of the probes is arbitrary, and thus the contents of thefirst probe and/or second probe as disclosed herein are interchangeable.

In some embodiments, the second probe includes ribonucleotides,deoxyribonucleotides, and/or synthetic nucleotides that are capable ofparticipating in Watson-Crick type or analogous base pair interactions.In some embodiments, the second probe includes deoxyribonucleotides. Insome embodiments, the second probe includes deoxyribonucleotides andribonucleotides. In some embodiments, the second probe includes adeoxyribonucleic acid that hybridizes to an analyte and includes aportion of the oligonucleotide that is not a deoxyribonucleic acid. Forexample, in some embodiments, the portion of the second probe that isnot a deoxyribonucleic acid is a ribonucleic acid or any othernon-deoxyribonucleic acid nucleic acid as described herein. In someembodiments where the second probe includes deoxyribonucleotides,hybridization of the second probe to the mRNA molecule results in aDNA:RNA hybrid. In some embodiments, the second probe includes onlydeoxyribonucleotides and upon hybridization of the first probe to themRNA molecule results in a DNA:RNA hybrid.

In some embodiments, the method includes a second probe that includesone or more sequences that are substantially complementary to one ormore sequences of an analyte. In some embodiments, a second probeincludes a sequence that is substantially complementary to a secondtarget sequence in the analyte. In some embodiments, the sequence of thesecond probe that is substantially complementary to the second targetsequence in the analyte is at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the second target sequence in theanalyte.

In some embodiments, a second probe includes a sequence that is about 10nucleotides to about 100 nucleotides (e.g., a sequence of about 10nucleotides to about 90 nucleotides, about 10 nucleotides to about 80nucleotides, about 10 nucleotides to about 70 nucleotides, about 10nucleotides to about 60 nucleotides, about 10 nucleotides to about 50nucleotides, about 10 nucleotides to about 40 nucleotides, about 10nucleotides to about 30 nucleotides, about 10 nucleotides to about 20nucleotides, about 20 nucleotides to about 100 nucleotides, about 20nucleotides to about 90 nucleotides, about 20 nucleotides to about 80nucleotides, about 20 nucleotides to about 70 nucleotides, about 20nucleotides to about 60 nucleotides, about 20 nucleotides to about 50nucleotides, about 20 nucleotides to about 40 nucleotides, about 20nucleotides to about 30 nucleotides, about 30 nucleotides to about 100nucleotides, about 30 nucleotides to about 90 nucleotides, about 30nucleotides to about 80 nucleotides, about 30 nucleotides to about 70nucleotides, about 30 nucleotides to about 60 nucleotides, about 30nucleotides to about 50 nucleotides, about 30 nucleotides to about 40nucleotides, about 40 nucleotides to about 100 nucleotides, about 40nucleotides to about 90 nucleotides, about 40 nucleotides to about 80nucleotides, about 40 nucleotides to about 70 nucleotides, about 40nucleotides to about 60 nucleotides, about 40 nucleotides to about 50nucleotides, about 50 nucleotides to about 100 nucleotides, about 50nucleotides to about 90 nucleotides, about 50 nucleotides to about 80nucleotides, about 50 nucleotides to about 70 nucleotides, about 50nucleotides to about 60 nucleotides, about 60 nucleotides to about 100nucleotides, about 60 nucleotides to about 90 nucleotides, about 60nucleotides to about 80 nucleotides, about 60 nucleotides to about 70nucleotides, about 70 nucleotides to about 100 nucleotides, about 70nucleotides to about 90 nucleotides, about 70 nucleotides to about 80nucleotides, about 80 nucleotides to about 100 nucleotides, about 80nucleotides to about 90 nucleotides, or about 90 nucleotides to about100 nucleotides).

In some embodiments, a sequence of the second probe that issubstantially complementary to a sequence in the analyte includes asequence that is about 5 nucleotides to about 50 nucleotides (e.g.,about 5 nucleotides to about 45 nucleotides, about 5 nucleotides toabout 40 nucleotides, about 5 nucleotides to about 35 nucleotides, about5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 25nucleotides, about 5 nucleotides to about 20 nucleotides, about 5nucleotides to about 15 nucleotides, about 5 nucleotides to about 10nucleotides, about 10 nucleotides to about 50 nucleotides, about 10nucleotides to about 45 nucleotides, about 10 nucleotides to about 40nucleotides, about 10 nucleotides to about 35 nucleotides, about 10nucleotides to about 30 nucleotides, about 10 nucleotides to about 25nucleotides, about 10 nucleotides to about 20 nucleotides, about 10nucleotides to about 15 nucleotides, about 15 nucleotides to about 50nucleotides, about 15 nucleotides to about 45 nucleotides, about 15nucleotides to about 40 nucleotides, about 15 nucleotides to about 35nucleotides, about 15 nucleotides to about 30 nucleotides, about 15nucleotides to about 25 nucleotides, about 15 nucleotides to about 20nucleotides, about 20 nucleotides to about 50 nucleotides, about 20nucleotides to about 45 nucleotides, about 20 nucleotides to about 40nucleotides, about 20 nucleotides to about 35 nucleotides, about 20nucleotides to about 30 nucleotides, about 20 nucleotides to about 25nucleotides, about 25 nucleotides to about 50 nucleotides, about 25nucleotides to about 45 nucleotides, about 25 nucleotides to about 40nucleotides, about 25 nucleotides to about 35 nucleotides, about 25nucleotides to about 30 nucleotides, about 30 nucleotides to about 50nucleotides, about 30 nucleotides to about 45 nucleotides, about 30nucleotides to about 40 nucleotides, about 30 nucleotides to about 35nucleotides, about 35 nucleotides to about 50 nucleotides, about 35nucleotides to about 45 nucleotides, about 35 nucleotides to about 40nucleotides, about 40 nucleotides to about 50 nucleotides, about 40nucleotides to about 45 nucleotides, or about 45 nucleotides to about 50nucleotides).

In some embodiments, a second probe includes a capture probe capturedomain sequence. As used herein, a “capture probe capture domain” is asequence, domain, or moiety that can bind specifically to a capturedomain of a capture probe. In some embodiments, “capture domain capturedomain” can be used interchangeably with “capture probe binding domain.”In some embodiments, a second probe includes a sequence from 5′ to 3′: asequence that is substantially complementary to a sequence in theanalyte and a capture probe capture domain.

In some embodiments, a capture probe capture domain includes a poly(A)sequence. In some embodiments, the capture probe capture domain includesa poly-uridine sequence, a poly-thymidine sequence, or both. In someembodiments, the capture probe capture domain includes a random sequence(e.g., a random hexamer or octamer). In some embodiments, the captureprobe capture domain is complementary to a capture domain in a captureprobe that detects a particular target(s) of interest. In someembodiments, a capture probe capture domain blocking moiety thatinteracts with the capture probe capture domain is provided. In someembodiments, a capture probe capture domain blocking moiety includes asequence that is complementary or substantially complementary to acapture probe capture domain. In some embodiments, a capture probecapture domain blocking moiety prevents the capture probe capture domainfrom binding the capture probe when present. In some embodiments, acapture probe capture domain blocking moiety is removed prior to bindingthe capture probe capture domain (e.g., present in a ligated probe) to acapture probe. In some embodiments, a capture probe capture domainblocking moiety includes a poly-uridine sequence, a poly-thymidinesequence, or both. In some embodiments, the capture probe capture domainsequence includes ribonucleotides, deoxyribonucleotides, and/orsynthetic nucleotides that are capable of participating in Watson-Cricktype or analogous base pair interactions. In some embodiments, thecapture probe binding domain sequence includes at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, thecapture probe binding domain sequence includes at least 25, 30, or 35nucleotides.

In some embodiments, a second probe includes a phosphorylated nucleotideat the 5′ end. The phosphorylated nucleotide at the 5′ end can be usedin a ligation reaction to ligate the second probe to the first probe.

As shown in FIG. 6, a non-limiting example of a second probe 604, whichcan be referred to a RHS probe, includes a sequence 605 that issubstantially complementary to a second target sequence on the analyte607 and a capture probe capture domain 606.

In some embodiments, a second probe includes an auxiliary sequence thatdoes not hybridize to an analyte. In some embodiments, the auxiliarysequence can be used to hybridize to additional probes.

(iii) Multiple Probes

In some embodiments, the methods of target RNA capture as disclosedherein include multiple probe oligonucleotides. In some embodiments, themethods include 2, 3, 4, or more probe oligonucleotides. In someembodiments, each of the probe oligonucleotides includesribonucleotides, deoxyribonucleotides, and/or synthetic nucleotides thatare capable of participating in Watson-Crick type or analogous base pairinteractions. In some embodiments, each of the probe oligonucleotidesincludes deoxyribonucleotides. In some embodiments, each of the probeoligonucleotides includes deoxyribonucleotides and ribonucleotides.

In some instances, the multiple probes span different target sequences,and multiple, serial ligation steps are carried out to determine thelocation and abundance of an analyte.

In some instances, the methods include a first probe and multiple secondprobes (or vice versa) are used, with the multiple second probeshybridizing to different sequences (e.g., wild-type versus mutantsequence, different isoforms, splice variants) in order to identify thesequence of an analyte. It is appreciated that this method can beutilized to detect single mutations (e.g., point mutations, SNPs, splicevariants, etc.) or can multi-nucleotide mutations (e.g., insertions,deletions, etc.).

Methods provided herein may be applied to a single nucleic acid moleculeor a plurality of nucleic acid molecules. A method of analyzing a samplecomprising a nucleic acid molecule may comprise providing a plurality ofnucleic acid molecules (e.g., RNA molecules), where each nucleic acidmolecule comprises a first target region (e.g., a first target sequence)and a second target region (e.g., a second target sequence), a pluralityof first probe oligonucleotides, and a plurality of second probeoligonucleotides. In some cases, one or more target regions of nucleicacid molecules of the plurality of nucleic acid molecules may comprisethe same sequence. The first and second target regions (e.g., the firstand second target sequences) of a nucleic acid molecule of the pluralityof nucleic acid molecules may be adjacent to one another.

(iv) First Probe Having a Linker Sequence

Also provided herein are methods for identifying a location of ananalyte in a biological sample where the method includes a first probethat includes a linker and a second probe. Using a pair of probes wherethe first probe includes a linker sequence enables greater flexibilityin designing RTL probes, primarily by increasing the sequences withinthe analyte that can be used as optional target sequences.

As used herein, a “linker sequence” can refer to one or more nucleicacids sequences on a probe (e.g., a first probe, a second probe, or aspanning probe that are disposed between sequences that hybridize to ananalyte, sequences that link together the analyte specific sequences ofa probe). In some embodiments, a linker includes a sequence that is notsubstantially complementary to either the sequence of the target analyteor to the analyte specific sequences of a first probe, a second probe,or a spanning probe. In some embodiments, the linker sequence includesribonucleotides, deoxyribonucleotides, and/or synthetic nucleotides,where the sequence within the linker is not substantially complementaryto the target analyte or the analyte specific sequences of a firstprobe, a second probe, or a spanning probe.

In some embodiments where a first and/or a second probe include a linkersequence, the linker sequence can include a total of about 10nucleotides to about 100 nucleotides, or any of the subranges describedherein.

In some embodiments, a linker sequence includes a barcode sequence thatserves as a proxy for identifying the analyte. In some embodiments, thebarcode sequence is a sequence that is at least 70% identical (e.g., atleast 75% identical, at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, or at least 99% identical)to a sequence in the analyte. In some embodiments where a linkersequence includes a barcode sequence, the barcode sequence is located 5′to the linker sequence. In some embodiments where a linker sequenceincludes a barcode sequence, the barcode sequence is located 3′ to thelinker sequence. In some embodiments, the barcode sequence is disposedbetween two linker sequences. In such cases, the two linker sequencesflanking the barcode sequence can be considered to be a part of the samelinker sequence.

In some embodiments where a first and/or a second probe include a linkersequence, the linker sequence can include ribonucleotides,deoxyribonucleotides, and/or synthetic nucleotides.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample includes a first probe that includes alinker sequence and a second probe comprising: (a) contacting thebiological sample with a substrate including a plurality of captureprobes, wherein a capture probe of the plurality of capture probesincludes a capture domain and a spatial barcode; (b) contacting thebiological sample with a first probe and a second probe, wherein aportion of the first probe and a portion of the second probe aresubstantially complementary to adjacent sequences of the analyte,wherein the first probe includes: (i) a first sequence that issubstantially complementary to a first target sequence of the analyte;(ii) a linker sequence; (iii) a second sequence that is substantiallycomplementary to a second target sequence of the analyte; and whereinthe second probe includes a sequence that is substantially complementaryto a third target sequence of the analyte and a capture probe capturedomain that is capable of binding to a capture domain of a captureprobe; (c) hybridizing the first probe and the second probe to theanalyte; (d) ligating the first probe and the second probe, therebycreating a ligation product; (e) releasing the ligation product from theanalyte; (f) hybridizing the capture probe binding domain to a capturedomain; and (g) determining (i) all or a part of the sequence of theligation product specifically bound to the capture domain, or acomplement thereof, and (ii) all or a part of the sequence of thespatial barcode, or a complement thereof, and using the determinedsequence of (i) and (ii) to identify the location of the analyte in thebiological sample.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample where the method includes a first probethat includes a linker and a second probe can include the components asshown FIG. 8. A first probe 801 includes a functional sequence 802, afirst sequence 803 that sequence that is substantially complementary toa first target sequence 804 of the analyte, a linker sequence 805; and asecond sequence 806 that is substantially complementary to a secondtarget sequence 807 of the analyte. A second probe 808 includes asequence 809 that is substantially complementary to a third targetsequence 810 of the analyte and a capture probe capture domain 811 thatis capable of binding to a capture domain of a capture probe.

1) First Probe

In some embodiments where first probe includes a linker sequence, thefirst probe includes a first sequence that is substantiallycomplementary to a first target sequence of the analyte, a linkersequence, and a second sequence that is substantially complementary tosecond target sequence of the analyte. In some embodiments, a firstprobe includes from 5′ to 3′: a first sequence that is substantiallycomplementary to a first target sequence of the analyte, a linkersequence, and a second sequence that is substantially complementary tosecond target sequence of the analyte.

In some embodiments where a first probe includes a linker sequence, thefirst probe includes a functional sequence. In some embodiments, a firstprobe includes a functional sequence, a first sequence that issubstantially complementary to a first target sequence of the analyte, alinker sequence, and a second sequence that is substantiallycomplementary to second target sequence of the analyte. In someembodiments, the functional sequence includes a primer sequence.

In some embodiments where a first probe includes a linker sequence, afirst probe includes at least two ribonucleic acid bases at the 3′ end.In some embodiments, a first probe includes at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten ribonucleic acid bases at the 3′ end.

In some embodiments where a first probe includes a linker, the firstprobe includes a sequence that is about 10 nucleotides to about 300nucleotides (e.g., a sequence of about 10 nucleotides to about 300nucleotides, about 10 nucleotides to about 250 nucleotides, about 10nucleotides to about 200 nucleotides, about 10 nucleotides to about 150nucleotides, about 10 nucleotides to about 100 nucleotides, about 10nucleotides to about 50 nucleotides, about 50 nucleotides to about 300nucleotides, about 50 nucleotides to about 250 nucleotides, about 50nucleotides to about 200 nucleotides, about 50 nucleotides to about 150nucleotides, about 50 nucleotides to about 100 nucleotides, about 100nucleotides to about 300 nucleotides, about 100 nucleotides to about 250nucleotides, about 100 nucleotides to about 200 nucleotides, about 100nucleotides to about 150 nucleotides, about 150 nucleotides to about 300nucleotides, about 150 nucleotides to about 250 nucleotides, about 150nucleotides to about 200 nucleotides, about 200 nucleotides to about 300nucleotides, about 200 nucleotides to about 250 nucleotides, or about250 nucleotides to about 300 nucleotides).

In some embodiments where a first probe includes a linker sequence, thefirst probe includes a first sequence that is substantiallycomplementary to a first target sequence of the analyte. In someembodiments, the first sequence of the first probe is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to thefirst target sequence in the analyte. In some embodiments, the firstsequence of the first probe that is substantially complementary to afirst target sequence can include a sequence that is about 5 nucleotidesto about 50 nucleotides, or any of the subranges described herein.

In some embodiments where a first probe includes a linker, the firstprobe includes a second sequence that is substantially complementary toa second target sequence of the analyte. In some embodiments, the secondsequence of the first probe is at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the second target sequence in theanalyte. In some embodiments, the second sequence of the first probethat is substantially complementary to a second target sequence caninclude a sequence that is about 5 nucleotides to about 50 nucleotides,or any of the subranges described herein.

In some embodiments, a first probe that includes a linker sequenceincludes ribonucleotides, deoxyribonucleotides, and/or syntheticnucleotides that are capable of participating in Watson-Crick type oranalogous base pair interactions. In some embodiments, the first probethat includes a linker sequence includes deoxyribonucleotides. In someembodiments, the first probe that includes a linker sequence includesdeoxyribonucleotides and ribonucleotides. In some embodiments where thefirst probe that includes a linker sequence includesdeoxyribonucleotides, hybridization of the first probe to the mRNAmolecule results in a DNA:RNA hybrid. In some embodiments, the firstprobe that includes a linker sequence includes only deoxyribonucleotidesand upon hybridization of the first probe to the mRNA molecule resultsin a DNA:RNA hybrid.

2) Second Probe

In some embodiments where a first probe includes a linker sequence, asecond probe includes a sequence that is substantially complementary toa third target sequence of the analyte and a capture probe capturedomain that is capable of binding to a capture domain of a captureprobe. In some embodiments where a first probe includes a linker, asecond probe includes a sequence that is about 10 nucleotides to about100 nucleotides, or any of the subranges described herein.

In some embodiments where a first probe includes a linker, the sequenceof the second probe is at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to the third target sequence in theanalyte. In some embodiments, the sequence of the second probe that issubstantially complementary to a third target sequence can include asequence that is about 5 nucleotides to about 50 nucleotides, or any ofthe subranges described herein.

In some embodiments where a first probe includes a linker sequence, afirst target sequence is not adjacent to a second target sequence. Forexample, the first target sequence and second target sequences arelocated on different exons of the same mRNA molecule. In anotherexample, the first target sequence and the second target sequence arelocated on the same exon of the same mRNA molecule but are not adjacent.In some instances, the first probe and the second probe hybridize tosequences that at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotidesapart.

In some embodiments where a first probe includes a linker sequence, asecond target sequence is directly adjacent to a third target sequence.

In some embodiments where a first probe includes a linker sequence, thesecond probe includes ribonucleotides, deoxyribonucleotides, and/orsynthetic nucleotides that are capable of participating in Watson-Cricktype or analogous base pair interactions. In some embodiments, thesecond probe includes deoxyribonucleotides. In some embodiments, thesecond probe includes deoxyribonucleotides and ribonucleotides. In someembodiments where the second probe includes deoxyribonucleotides,hybridization of the second probe to the mRNA molecule results in aDNA:RNA hybrid. In some embodiments, the second probe includes onlydeoxyribonucleotides and upon hybridization of the second probe to themRNA molecule results in a DNA:RNA hybrid.

(v) Second Probe Having a Linker

Also provided herein are methods for identifying a location of ananalyte in a biological sample where the method includes a first probeand a second probe that includes a linker sequence. Using a pair ofprobes where the second probe includes a linker sequence enables greaterflexibility in designing RTL probes, primarily by increasing thesequences within the analyte that can be used as optional targetsequences.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample where the method includes a first probeand a second probe that includes a linker includes: (a) contacting thebiological sample with a substrate including a plurality of captureprobes, wherein a capture probe of the plurality of capture probesincludes a capture domain and a spatial barcode; (b) contacting thebiological sample with a first probe and a second probe, wherein aportion of the first probe and a portion of the second probe aresubstantially complementary to adjacent sequences of the analyte,wherein the first probe includes a sequence that is substantiallycomplementary to a first target sequence of the analyte, wherein thesecond probe includes: (i) a first sequence that is substantiallycomplementary to a second target sequence of the analyte; (ii) a linkersequence; (iii) a second sequence that is substantially complementary toa third target sequence of the analyte; and (iv) a capture probe bindingdomain that is capable of binding to a capture domain of a captureprobe; (c) hybridizing the first probe and the second probe to theanalyte; (d) ligating the first probe and the second probe, therebycreating a ligation product; (e) releasing the ligation product from theanalyte; (f) hybridizing the capture probe binding domain to a capturedomain; and (g) determining (i) all or a part of the sequence of theligation product specifically bound to the capture domain, or acomplement thereof, and (ii) all or a part of the sequence of thespatial barcode, or a complement thereof, and using the determinedsequence of (i) and (ii) to identify the location of the analyte in thebiological sample.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample where the method includes a first probeand a second probe that includes a linker can include the components asshown FIG. 9. A first probe 901 includes a functional sequence 902 and asequence 903 that is substantially complementary to a first targetsequence 904 of the analyte. A second probe 905 includes a firstsequence 906 that is substantially complementary to a second targetsequence 907 of the analyte, a linker sequence 908; and a secondsequence 909 that is substantially complementary to a second targetsequence 910 of the analyte, and a capture probe capture domain 911 thatis capable of binding to a capture domain of a capture probe.

1) First Probe

In some embodiments where a second probe includes a linker sequence, afirst probe includes a sequence that is substantially complementary to afirst target sequence of the analyte.

In some embodiments where a second probe includes a linker sequence, afirst probe includes a functional sequence. In some embodiments, a firstprobe includes a functional sequence and a sequence that issubstantially complementary to a first target sequence of the analyte.In some embodiments, the functional sequence includes a primer sequence.In some embodiments, the first probe oligonucleotide includes from 5′ to3′: a functional sequence, and a sequence that is substantiallycomplementary to a first target sequence.

In some embodiments where a linker is on a second probe, a first probeincludes a sequence that is about 10 nucleotides to about 100nucleotides, or any of the subranges described herein.

In some embodiments where the second probe includes a linker sequence, asequence of a first probe that is substantially complementary to a firsttarget sequence of the analyte is at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% complementary to the first target sequence inthe analyte. In some embodiments, the sequence that is substantiallycomplementary to a first target sequence can include a sequence that isabout 5 nucleotides to about 50 nucleotides, or any of the subrangesdescribed herein.

In some embodiments where a second probe includes a linker sequence, afirst probe includes at least two ribonucleic acid bases at the 3′ end.In some embodiments, a first probe includes at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten ribonucleic acid bases at the 3′ end.

In some embodiments where a second probe includes a linker sequence, thefirst probe includes ribonucleotides, deoxyribonucleotides, and/orsynthetic nucleotides that are capable of participating in Watson-Cricktype or analogous base pair interactions. In some embodiments, the firstprobe includes deoxyribonucleotides. In some embodiments, the firstprobe includes deoxyribonucleotides and ribonucleotides. In someembodiments where the first probe includes deoxyribonucleotides,hybridization of the first probe to the mRNA molecule results in aDNA:RNA hybrid. In some embodiments, the first probe includes onlydeoxyribonucleotides and upon hybridization of the first probe to themRNA molecule results in a DNA:RNA hybrid.

2) Second Probe

In some embodiments where a linker is on a second probe, the secondprobe includes (i) a first sequence that is substantially complementaryto a second target sequence of the analyte; (ii) a linker sequence(e.g., any of the exemplary linker sequences described herein); (iii) asecond sequence that is substantially complementary to third targetsequence of the analyte; and (iv) a capture probe capture domain (e.g.,any of the exemplary capture probe capture domains described herein)that is capable of binding to a capture domain of a capture probe.

In some embodiments where a second probe includes a linker sequence, thesecond probe includes a sequence that is about 10 nucleotides to about300 nucleotides, or any of the subranges described herein.

In some embodiments where a second probe includes a linker sequence, thesecond probe includes a first sequence that is substantiallycomplementary to a second target sequence of the analyte. In someembodiments, the first sequence of the second probe is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to thesecond target sequence in the analyte. In some embodiments, the firstsequence of the second probe that is substantially complementary to asecond target sequence can include a sequence that is about 5nucleotides to about 50 nucleotides, or any of the subranges describedherein.

In some embodiments where a second probe includes a linker sequence, thesecond probe includes a second sequence that is substantiallycomplementary to a third target sequence of the analyte. In someembodiments, the second sequence of the second probe is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complementary to thethird target sequence in the analyte. In some embodiments, the secondsequence of the second probe that is substantially complementary to athird target sequence can include a sequence that is about 5 nucleotidesto about 50 nucleotides, or any of the subranges described herein.

In some embodiments where a second probe includes a linker sequence, asecond target sequence is not adjacent to a third target sequence in themRNA molecule. For example, the second target sequence and third targetsequence are located on different exons of the same mRNA molecule. Inanother example, the second target sequence and the third targetsequence are located on the same exon of the same mRNA molecule but arenot adjacent.

In some embodiments where a second probe includes a linker sequence, afirst target sequence is directly adjacent to a second target sequence.

In some embodiments, a second probe that includes a linker sequenceincludes ribonucleotides, deoxyribonucleotides, and/or syntheticnucleotides that are capable of participating in Watson-Crick type oranalogous base pair interactions. In some embodiments, the second probethat includes a linker sequence includes deoxyribonucleotides. In someembodiments, the second probe that includes a linker sequence includesdeoxyribonucleotides and ribonucleotides. In some embodiments where thesecond probe that includes a linker sequence includesdeoxyribonucleotides, hybridization of the second probe to the mRNAmolecule results in a DNA:RNA hybrid. In some embodiments, the secondprobe that includes a linker sequence includes only deoxyribonucleotidesand upon hybridization of the second probe to the mRNA molecule resultsin a DNA:RNA hybrid.

(vii) Probe Combination with Linkers on the First Probe and the SecondProbe

Also provided herein are methods for identifying a location of ananalyte in a biological sample where the method includes a first probethat includes a linker sequence and a second probe that includes alinker sequence. Using a pair of probes where the first probe and secondprobe each include a linker sequence enables greater flexibility indesigning RTL probes, primarily by increasing the sequences within theanalyte that can be used as optional target sequences.

(c) Probe Combinations Including a First Probe, a Second Probe and aSpanning Probe

Also provided herein are methods for identifying a location of ananalyte in a biological sample where the method includes a first probe,a spanning probe, and a second probe. Using a spanning probe enablesgreater flexibility in designing RTL probes, primarily by increasing thesequences within the analyte that can be used as optional targetsequences. In some cases, using a spanning probe can also be used tointerrogate the variants (e.g., splice variants) that span greaterdistances that can be interrogated using a first or second probe with alinker sequence.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample where the method includes a first probe,a spanning probe, and a second probe, includes: (a) contacting thebiological sample with a substrate including a plurality of captureprobes, wherein a capture probe of the plurality of capture probesincludes a capture domain and a spatial barcode; (b) contacting thebiological sample with a first probe, a second probe, and one or morespanning probes, wherein the first probe is substantially complementaryto a first portion of the analyte, wherein the second probe issubstantially complementary to a second portion of the analyte andfurther includes a capture probe binding domain, and wherein thespanning probe includes: (i) a first sequence that is substantiallycomplementary to a first target sequence of the analyte, and (ii) asecond sequence that is substantially complementary to a second targetsequence of the analyte; (c) hybridizing the first probe, the secondprobe, and the spanning probe to the analyte; (d) ligating the firstprobe, the one or more spanning probes, and the second probe, therebycreating a ligation product that is substantially complementary to theanalyte; (e) releasing the ligation product from the analyte; (f)hybridizing the capture probe binding domain to a capture domain; and(g) determining (i) all or a part of the sequence of the ligationproduct specifically bound to the capture domain, or a complementthereof, and (ii) all or a part of the sequence of the spatial barcode,or a complement thereof, and using the determined sequence of (i) and(ii) to identify the location of the analyte in the biological sample.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample where the method includes a first probe,a second probe, and a spanning probe can include the components as shownFIG. 10. A first probe 1001 includes a functional sequence 1002, asequence 1003 that is substantially complementary to a first portion1004 of the analyte. A spanning probe 1005 includes a first sequence1006 that is substantially complementary to a first target sequence 1007of the analyte, a linker sequence 1008, and a second sequence 1009 thatis substantially complementary to a second target sequence 1010 of theanalyte. A second probe 1011 includes a sequence 1012 that issubstantially complementary to a second portion 1013 of the analyte anda capture probe capture domain 1014.

(i) First Probe

In some embodiments where the method includes a spanning probe, a firstprobe includes a sequence that is substantially complementary to a firstportion of the analyte. In some embodiments, a sequence of the firstprobe is at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% complementary to a first portion of the analyte. In someembodiments, the sequence of the first probe that is substantiallycomplementary to a first portion of the analyte can include a sequencethat is about 5 nucleotides to about 50 nucleotides, or any of thesubranges described herein. In some embodiments, the first probeincludes a functional sequence. In some embodiments, the functionalsequence is a primer sequence.

In some embodiments where the method includes a spanning probe, a firstprobe includes at least two ribonucleic acid bases at the 3′ end. Insome embodiments, a first probe includes at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, or at least ten ribonucleic acid bases at the 3′ end.

In some embodiments where the method includes a spanning probe, a firstprobe includes from 5′ to 3′: a functional sequence, a sequence that issubstantially complementary to a first portion of the analyte, and twoor more ribonucleic acid bases.

In some embodiments where the method includes a spanning probe, a firstprobe includes ribonucleotides, deoxyribonucleotides, and/or syntheticnucleotides that are capable of participating in Watson-Crick type oranalogous base pair interactions. In some embodiments, the first probeincludes deoxyribonucleotides. In some embodiments, the first probeincludes deoxyribonucleotides and ribonucleotides. In some embodimentswhere the first probe includes deoxyribonucleotides, hybridization ofthe first probe to the mRNA molecule results in a DNA:RNA hybrid. Insome embodiments, the first probe includes only deoxyribonucleotides andupon hybridization of the first probe to the mRNA molecule results in aDNA:RNA hybrid.

(ii) Spanning Probe

In some embodiments where the method includes a spanning probe, thespanning probe includes a first sequence that is substantiallycomplementary to a first target sequence of the analyte, and a secondsequence that is substantially complementary to a second target sequenceof the analyte. In some embodiments, the spanning probe includes a firstsequence that is substantially complementary to a first target sequenceof the analyte, a functional sequence, and a second sequence that issubstantially complementary to a second target sequence of the analyte.In some embodiments, a spanning probe includes from 5′ to 3′: a firstsequence, a functional sequence, and a second sequence. In someembodiments, the functional sequence is a linker sequence. The linkersequence can include a total of about 10 nucleotides to about 100nucleotides, or any of the subranges described herein.

In some embodiments, the functional sequence includes a barcodesequence. In some embodiments, the spanning probe can include a linkerand a barcode sequence. In such cases, linker sequences can flank thebarcode, the barcode can be 5′ to a linker sequence, or the barcode canbe 3′ to a linker sequence. In some embodiments, a barcode sequence isflanked by a 5′ linker sequence (e.g., any of the exemplary linkersequences described herein) and a 3′ linker sequence (e.g., any of theexemplary linker sequences described herein).

In some embodiments, the spanning probe includes from 5′ to 3′: a firstsequence, a 5′ linker sequence, a barcode, a 3′ linker sequence, and asecond sequence.

In some embodiments, the spanning probes includes a sequence that isabout 10 nucleotides to about 300 nucleotides or any of the subrangesdescribed herein.

In some embodiments, a first sequence of the spanning probe is at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% complementary tothe first target sequence of the analyte. In some embodiments, a secondsequence of the spanning probe is at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% complementary to the second target sequenceof the analyte.

In some embodiments, the first sequence of the spanning probe and thesecond sequence of the spanning probe are substantially complementary tosequences within the same exon.

In some embodiments, the first target sequence of the analyte and thesecond target of the analyte are located within the same exon. In suchcases, the first target sequence and the second target sequence are notdirectly adjacent.

In some embodiments, the first sequence of the spanning probe and thesecond sequence of the spanning probe are substantially complementary tosequences within the different exons of the same gene. In someembodiments, the first target sequence of the analyte and the secondtarget sequence of the analyte are located on different exons of thesame gene.

In some embodiments, the spanning probe includes ribonucleotides,deoxyribonucleotides, and/or synthetic nucleotides that are capable ofparticipating in Watson-Crick type or analogous base pair interactions.In some embodiments, the spanning probe includes deoxyribonucleotides.In some embodiments, the spanning probe includes deoxyribonucleotidesand ribonucleotides. In some embodiments where the spanning probeincludes deoxyribonucleotides, hybridization of the spanning probe tothe mRNA molecule results in a DNA:RNA hybrid. In some embodiments, thespanning probe includes only deoxyribonucleotides and upon hybridizationof the spanning probe to the mRNA molecule results in a DNA:RNA hybrid.

(iii) Second Probe

In some embodiments where the method includes a spanning probe, a secondprobe includes a sequence that is substantially complementary to asecond portion of the analyte and a capture probe capture domain (e.g.,any of the exemplary capture probe capture domains described herein).

In some embodiments where the method includes a spanning probe, asequence of the second probe is at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% complementary to a second portion of theanalyte. In some embodiments, the sequence of the second probe that issubstantially complementary to a second portion of the analyte caninclude a sequence that is about 5 nucleotides to about 50 nucleotides,or any of the subranges described herein.

In some embodiments where the method includes a spanning probe, thefirst portion of the analyte is directly adjacent to the first targetsequence, and/or the second portion of the analyte is directly adjacentto the second target sequence. In such cases, the sequence of the firstprobe is ligated to the first sequence of the spanning probe, and thesequence of the second probe is ligated to the second sequence of thespanning probe. In some embodiments, the spanning probe includes atleast two ribonucleic acid based at the 3′ end, the first probe includesat least two ribonucleic acids at the 3′ end, or both. In someembodiments, the spanning probe includes a phosphorylated nucleotide atthe 5′ end, the second probe includes a phosphorylated nucleotide at the5′ end, or both.

In some embodiments where the method includes a spanning probe, a secondprobe includes ribonucleotides, deoxyribonucleotides, and/or syntheticnucleotides that are capable of participating in Watson-Crick type oranalogous base pair interactions. In some embodiments, the second probeincludes deoxyribonucleotides. In some embodiments, the second probeincludes deoxyribonucleotides and ribonucleotides. In some embodimentswhere the second probe includes deoxyribonucleotides, hybridization ofthe second probe to the mRNA molecule results in a DNA:RNA hybrid. Insome embodiments, the second probe includes only deoxyribonucleotidesand upon hybridization of the second probe to the mRNA molecule resultsin a DNA:RNA hybrid.

(iv) Probe Combinations Including a First Probe, a Second Probe, andMultiple Spanning Probes

Also provided herein are methods for identifying a location of ananalyte in a biological sample where the method includes a first probe,at least two spanning probes, and a second probe. Using two or morespanning probes enables greater flexibility in designing RTL probes,primarily by increasing the sequences within the analyte that can beused as optional target sequences. In some cases, using two or morespanning probe can also be used to interrogate the variants (e.g.,splice variants) that span greater distances that can be interrogatedusing one spanning probe.

A non-limiting example of a method for identifying a location of ananalyte in a biological sample where the method includes a first probe,two or more spanning probes, and a second probe, includes: (a)contacting the biological sample with a substrate including a pluralityof capture probes, wherein a capture probe of the plurality of captureprobes includes a capture domain and a spatial barcode; (b) contactingthe biological sample with a first probe, a second probe, and twospanning probes, wherein the first probe is substantially complementaryto a first portion of the analyte, wherein the second probe issubstantially complementary to a second portion of the analyte andfurther includes a capture probe binding domain, and wherein the firstspanning probe includes: (i) a first sequence that is substantiallycomplementary to a first target sequence of the analyte, and (ii) asecond sequence that is substantially complementary to a second targetsequence of the analyte; and the second spanning probe includes (i) athird sequence that is substantially complementary to a third targetsequence of the analyte, and (ii) a fourth sequence that issubstantially complementary to a fourth target sequence of the analyte;(c) hybridizing the first probe, the second probe, and the spanningprobe to the analyte; (d) ligating the first probe, the one or morespanning probes, and the second probe, thereby creating a ligationproduct that is substantially complementary to the analyte; (e)releasing the ligation product from the analyte; (f) hybridizing thecapture probe binding domain to a capture domain; and (g) determining(i) all or a part of the sequence of the ligation product specificallybound to the capture domain, or a complement thereof, and (ii) all or apart of the sequence of the spatial barcode, or a complement thereof,and using the determined sequence of (i) and (ii) to identify thelocation of the analyte in the biological sample.

In some embodiments, the methods that include one or more spanningprobes include at least two, at least three, at least four, at leastfive, or more spanning probes. In such cases, the one or more spanningprobes includes (i) a third sequence that is substantially complementaryto a third target sequence of the analyte, and (ii) a fourth sequencethat is substantially complementary to a fourth target sequence of theanalyte.

In some embodiments where the method includes two (or more) spanningprobes, the first target sequence is located in a first exon, the secondtarget sequence is located in a second exon, and the third targetsequence and the fourth target sequence are located in a third exon. Insome embodiments where the method includes two (or more) spanningprobes, the first target sequence is located in a first exon, the secondtarget sequence is located in a second exon, and the third targetsequence is located in a third exon, and the fourth target sequence islocated in a fourth exon. In some embodiments where the method includestwo (or more) spanning probes, the first target sequence and the secondtarget sequences are located in a first exon, and the third targetsequence and the fourth target sequence are located in a second exon. Insome embodiments where the method includes two (or more) spanningprobes, the first target sequence and the second target sequences arelocated in a first exon, and the third target sequence is located in asecond exon, and the fourth target sequence is located in a third exon.

In some embodiments, where the methods include two (or more) spanningprobes, the method includes ligating: the first probe to the spanningprobe, the spanning probe to the one or more additional spanning probes,and the one or more additional spanning probes spanning oligonucleotideto the second probe, thereby creating a ligation product that includesone or more sequences that are substantially complementary to theanalyte. In some embodiments, where the methods include two (or more)spanning probes, the method includes ligating: the first probe to theone or more additional spanning probes, the one or more additionalspanning probes to the spanning probe, and the spanning probe to thesecond probe, thereby creating a ligation product that includes one ormore sequences that are substantially complementary to the analyte.

In some embodiments, each additional spanning probe can include afunctional sequence (e.g., any of the functional sequence describedherein). For example, each additional spanning probe can include alinker sequence (e.g., any of the exemplary linker sequences describedherein). In another example, each additional spanning probe can includea barcode sequence (e.g., any of the exemplary barcode sequencesdescribed herein) and a linker sequence (e.g., any of the linkersequences described herein). In some embodiments where an additionalspanning probe includes a barcode and a linker, a linker sequences canflank the barcode, the barcode can be 5′ to a linker sequence, or thebarcode can be 3′ to a linker sequence. In some embodiments, a barcodesequence is flanked by a 5′ linker sequence (e.g., any of the exemplarylinker sequences described herein) and a 3′ linker sequence (e.g., anyof the exemplary linker sequences described herein). In someembodiments, an additional spanning probe can include from 5′ to 3′: afirst sequence, a 5′ linker sequence, a barcode, a 3′ linker sequence,and a second sequence.

(d) Pre-Hybridization Methods

(i) Imaging and Staining

Prior to addition of the probes, in some instances, biological samplescan be stained using a wide variety of stains and staining techniques.In some instances, the biological sample is a section on a slide (e.g.,a 10 μm section). In some instances, the biological sample is driedafter placement onto a glass slide. In some instances, the biologicalsample is dried at 42° C. In some instances, drying occurs for about 1hour, about 2, hours, about 3 hours, or until the sections becometransparent. In some instances, the biological sample can be driedovernight (e.g., in a desiccator at room temperature).

In some embodiments, a sample can be stained using any number ofbiological stains, including but not limited to, acridine orange,Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin,ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine,methyl green, methylene blue, neutral red, Nile blue, Nile red, osmiumtetroxide, propidium iodide, rhodamine, or safranin. In some instances,the methods disclosed herein include imaging the biological sample. Insome instances, imaging the sample occurs prior to deaminating thebiological sample. In some instances, the sample can be stained usingknown staining techniques, including Can-Grunwald, Giemsa, hematoxylinand eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou,Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS)staining techniques. PAS staining is typically performed after formalinor acetone fixation. In some instances, the stain is an H&E stain.

In some embodiments, the biological sample can be stained using adetectable label (e.g., radioisotopes, fluorophores, chemiluminescentcompounds, bioluminescent compounds, and dyes) as described elsewhereherein. In some embodiments, a biological sample is stained using onlyone type of stain or one technique. In some embodiments, stainingincludes biological staining techniques such as H&E staining. In someembodiments, staining includes identifying analytes usingfluorescently-conjugated antibodies. In some embodiments, a biologicalsample is stained using two or more different types of stains, or two ormore different staining techniques. For example, a biological sample canbe prepared by staining and imaging using one technique (e.g., H&Estaining and brightfield imaging), followed by staining and imagingusing another technique (e.g., IHC/IF staining and fluorescencemicroscopy) for the same biological sample.

In some embodiments, biological samples can be destained. Methods ofdestaining or discoloring a biological sample are known in the art, andgenerally depend on the nature of the stain(s) applied to the sample.For example, H&E staining can be destained by washing the sample in HCl,or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid,benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid,succinic acid, salicylic acid, tartaric acid, sulfurous acid,trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid,orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citricacid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid,hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonicacid, hydrogen sulfide, or combinations thereof). In some embodiments,destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g.,HCl). In some embodiments, destaining can include adding HCl to adownstream solution (e.g., permeabilization solution). In someembodiments, destaining can include dissolving an enzyme used in thedisclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. Insome embodiments, after destaining hematoxylin with an acid, otherreagents can be added to the destaining solution to raise the pH for usein other applications. For example, SDS can be added to an aciddestaining solution in order to raise the pH as compared to the aciddestaining solution alone. As another example, in some embodiments, oneor more immunofluorescence stains are applied to the sample via antibodycoupling. Such stains can be removed using techniques such as cleavageof disulfide linkages via treatment with a reducing agent and detergentwashing, chaotropic salt treatment, treatment with antigen retrievalsolution, and treatment with an acidic glycine buffer. Methods formultiplexed staining and destaining are described, for example, inBolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin etal., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem.2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009;57:899-905, the entire contents of each of which are incorporated hereinby reference.

In some embodiments, immunofluorescence or immunohistochemistryprotocols (direct and indirect staining techniques) can be performed asa part of, or in addition to, the exemplary spatial workflows presentedherein. For example, tissue sections can be fixed according to methodsdescribed herein. The biological sample can be transferred to an array(e.g., capture probe array), wherein analytes (e.g., proteins) areprobed using immunofluorescence protocols. For example, the sample canbe rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X,1 U/μ1 RNAse inhibitor for 10 minutes at 4° C.) before being stainedwith fluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% TritonX, 1 U/μ1 RNAse inhibitor for 30 minutes at 4° C.). The biologicalsample can be washed, coverslipped (in glycerol+1 U/μ1 RNAse inhibitor),imaged (e.g., using a confocal microscope or other apparatus capable offluorescent detection), washed, and processed according to analytecapture or spatial workflows described herein.

In some instances, a glycerol solution and a cover slip can be added tothe sample. In some instances, the glycerol solution can include acounterstain (e.g., DAPI).

As used herein, an antigen retrieval buffer can improve antibody capturein IF/IHC protocols. An exemplary protocol for antigen retrieval can bepreheating the antigen retrieval buffer (e.g., to 95° C.), immersing thebiological sample in the heated antigen retrieval buffer for apredetermined time, and then removing the biological sample from theantigen retrieval buffer and washing the biological sample.

In some embodiments, optimizing permeabilization can be useful foridentifying intracellular analytes. Permeabilization optimization caninclude selection of permeabilization agents, concentration ofpermeabilization agents, and permeabilization duration. Tissuepermeabilization is discussed elsewhere herein.

In some embodiments, blocking an array and/or a biological sample inpreparation of labeling the biological sample decreases nonspecificbinding of the antibodies to the array and/or biological sample(decreases background). Some embodiments provide for blockingbuffers/blocking solutions that can be applied before and/or duringapplication of the label, wherein the blocking buffer can include ablocking agent, and optionally a surfactant and/or a salt solution. Insome embodiments, a blocking agent can be bovine serum albumin (BSA),serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk),casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), orpolyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidaseblocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodiumborohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blockingagent, and/or acetic acid. The blocking buffer/blocking solution can beapplied to the array and/or biological sample prior to and/or duringlabeling (e.g., application of fluorophore-conjugated antibodies) to thebiological sample.

(ii) Preparation of Sample for Application of Probes

In some instances, the biological sample is deparaffinized.Deparaffinization can be achieved using any method known in the art. Forexample, in some instances, the biological samples is treated with aseries of washes that include xylene and various concentrations ofethanol. In some instances, methods of deparaffinization includetreatment of xylene (e.g., three washes at 5 minutes each). In someinstances, the methods further include treatment with ethanol (e.g.,100% ethanol, two washes 10 minutes each; 95% ethanol, two washes 10minutes each; 70% ethanol, two washes 10 minutes each; 50% ethanol, twowashes 10 minutes each). In some instances, after ethanol washes, thebiological sample can be washed with deionized water (e.g., two washesfor 5 minutes each). It is appreciated that one skilled in the art canadjust these methods to optimize deparaffinization.

In some instances, the biological sample is decrosslinked. In someinstances, the biological sample is decrosslinked in a solutioncontaining TE buffer (comprising Tris and EDTA). In some instances, theTE buffer is basic (e.g., at a pH of about 9). In some instances,decrosslinking occurs at about 50° C. to about 80° C. In some instances,decrosslinking occurs at about 70° C. In some instances, decrosslinkingoccurs for about 1 hour at 70° C. Just prior to decrosslinking, thebiological sample can be treated with an acid (e.g., 0.1M HCl for about1 minute). After the decrosslinking step, the biological sample can bewashed (e.g., with 1×PBST).

In some instances, the methods of preparing a biological sample forprobe application include permeabilizing the sample. In some instances,the biological sample is permeabilized using a phosphate buffer. In someinstances, the phosphate buffer is PBS (e.g., 1×PBS). In some instances,the phosphate buffer is PBST (e.g., 1×PBST). In some instances, thepermeabilization step is performed multiple times (e.g., 3 times at 5minutes each).

In some instances, the methods of preparing a biological sample forprobe application include steps of equilibrating and blocking thebiological sample. In some instances, equilibrating is performed using apre-hybridization (pre-Hyb) buffer. In some instances, the pre-Hybbuffer is RNase-free. In some instances, the pre-Hyb buffer contains nobovine serum albumin (BSA), solutions like Denhardt's, or otherpotentially nuclease-contaminated biological materials.

In some instances, the equilibrating step is performed multiple times(e.g., 2 times at 5 minutes each; 3 times at 5 minutes each). In someinstances, the biological sample is blocked with a blocking buffer. Insome instances, the blocking buffer includes a carrier such as tRNA, forexample yeast tRNA such as from brewer's yeast (e.g., at a finalconcentration of 10-20 μg/mL). In some instances, blocking can beperformed for 5, 10, 15, 20, 25, or 30 minutes.

Any of the foregoing steps can be optimized for performance. Forexample, one can vary the temperature. In some instances, thepre-hybridization methods are performed at room temperature. In someinstances, the pre-hybridization methods are performed at 4° C. (in someinstances, varying the timeframes provided herein).

(e) Hybridizing the Probes

In some embodiments, the methods of targeted RNA capture provided hereininclude hybridizing a first probe oligonucleotide and a second probeoligonucleotide (e.g., a probe pair). In some instances, the first andsecond probe oligonucleotides each include sequences that aresubstantially complementary to one or more sequences (e.g., one or moretarget sequences) of an analyte of interest. In some embodiments, thefirst probe and the second probe bind to complementary sequences thatare completely adjacent (i.e., no gap of nucleotides) to one another orare on the same transcript.

In some instances, the methods include hybridization of probe sets,wherein the probe pairs are in a medium at a concentration of about 1 toabout 100 nM. In some instances, the concentration of the probe pairs isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, or 500 nM. In some instances, theconcentration of the probe pairs is 5 nM. In some instances, the probesets are diluted in a hybridization (Hyb) buffer. In some instances, theprobe sets are at a concentration of 5 nM in Hyb buffer.

In some instances, probe hybridization occurs at about 50° C. In someinstances, the temperature of probe hybridization ranges from about 30°C. to about 75° C., from about 35° C. to about 70° C., or from about 40°C. to about 65° C. In some embodiments, the temperature is about 30° C.,about 31° C., about 32° C., about 33° C., about 34° C., about 35° C.,about 36° C., about 37° C., about 38° C., about 39° C., about 40° C.,about 41° C., about 42° C., about 43° C., about 44° C., about 45° C.,about 46° C., about 47° C., about 48° C., about 49° C., about 50° C.,about 51° C., about 52° C., about 53° C., about 54° C., about 55° C.,about 56° C., about 57° C., about 58° C., about 59° C., about 60° C.,about 61° C., about 62° C., about 63° C., about 64° C., about 65° C.,about 66° C., about 67° C., about 68° C., about 69° C., or about 70° C.In some instances, probe hybridization occurs for about 30 minutes,about 1 hour, about 2 hours, about 2.5 hours, about 3 hours, or more. Insome instances, probe hybridization occurs for about 2.5 hours at 50° C.

In some instances, the hybridization buffer includes SSC (e.g., 1×SSC)or SSPE. In some instances, the hybridization buffer includes formamideor ethylene carbonate. In some instances, the hybridization bufferincludes one or more salts, like Mg salt for example MgCl₂, Na salt forexample NaCl, Mn salt for example MnCl₂. In some instances, thehybridization buffer includes Denhardt's solution, dextran sulfate,ficoll, PEG or other hybridization rate accelerators. In some instances,the hybridization buffer includes a carrier such as yeast tRNA, salmonsperm DNA, and/or lambda phage DNA. In some instances, the hybridizationbuffer includes one or more blockers. In some instances, thehybridization buffer includes RNase inhibitor(s). In some instances, thehybridization buffer can include BSA, sequence specific blockers,non-specific blockers, EDTA, RNase inhibitor(s), betaine, TMAC, or DMSO.In some instances, a hybridization buffer can further include detergentssuch as Tween, Triton-X 100, sarkosyl, and SDS. In some instances, thehybridization buffer includes nuclease-free water, DEPC water.

In some embodiments, the complementary sequences to which the firstprobe oligonucleotide and the second probe oligonucleotide bind are 1,2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95, about 100,about 125, about 150, about 175, about 200, about 250, about 300, about350, about 400, about 450, about 500, about 600, about 700, about 800,about 900, or about 1000 nucleotides away from each other. Gaps betweenthe probe oligonucleotides may first be filled prior to ligation, using,for example, Mu polymerase, DNA polymerase, RNA polymerase, reversetranscriptase, VENT polymerase, Taq polymerase, and/or any combinations,derivatives, and variants (e.g., engineered mutants) thereof. In someembodiments, when the first and second probe oligonucleotides areseparated from each other by one or more nucleotides, nucleotides areligated between the first and second probe oligonucleotides. In someembodiments, when the first and second probe oligonucleotides areseparated from each other by one or more nucleotides,deoxyribonucleotides are ligated between the first and second probeoligonucleotides.

In some instances, after hybridization, the biological sample is washedwith a post-hybridization wash buffer. In some instances, thepost-hybridization wash buffer includes one or more of SSC, yeast tRNA,formamide, ethylene carbonate, and nuclease-free water.

Additional embodiments regarding probe hybridization are furtherprovided.

(i) Hybridizing Temperatures

In some embodiments, the method described utilizes oligonucleotides thatinclude deoxyribonucleic acids (instead of strictly utilizingribonucleotides) at the site of ligation. Utilizing deoxyribonucleicacids in the methods described herein create a more uniform efficiencythat can be readily-controlled and flexible for various applications.

In a non-limiting example, the methods disclosed herein includecontacting a biological sample with a plurality of oligonucleotides(e.g., probes) including a first oligonucleotide (e.g., a first probe)and a second oligonucleotide (e.g., a second probe), wherein the firstoligonucleotide (e.g., the first probe) and the second oligonucleotide(e.g., the second probe) are complementary to a first sequence presentin an analyte and a second sequence present in the analyte,respectively; hybridizing the first oligonucleotide (e.g., the firstprobe) and the second oligonucleotide (e.g., the second probe) to theanalyte at a first temperature; hybridizing the first oligonucleotide(e.g., the first probe) and the second oligonucleotide (e.g., the secondprobe) to a third oligonucleotide (e.g., a splint oligonucleotide) at asecond temperature such that the first oligonucleotide (e.g., the firstprobe) and the second oligonucleotide (e.g., the second probe) abut eachother; ligating the first oligonucleotide (e.g., the first probe) to thesecond oligonucleotide (e.g., the second probe) to create a ligationproduct; contacting the biological sample with a substrate, wherein acapture probe is immobilized on the substrate, wherein the capture probeincludes a spatial barcode and a capture domain; allowing the ligationproduct to specifically bind to the capture domain; and determining (i)all or a part of the sequence of the ligation product specifically boundto the capture domain, or a complement thereof, and (ii) all or a partof the sequence of the spatial barcode, or a complement thereof, andusing the determined sequence of (i) and (ii) to identify the locationof the analyte in the biological sample; wherein the firstoligonucleotide (e.g., the first probe), the second oligonucleotide(e.g., the second probe), and the third oligonucleotide are DNAoligonucleotides, and wherein the first temperature is a highertemperature than the second temperature.

A non-limiting example of this method is shown in FIG. 11. A biologicalsample including an analyte 1101 is contacted with a first probe 1102and a second probe 1103. The first probe 1102 and the second probe 1103hybridize to the analyte at a first target sequence 1104 and a secondtarget sequence 1105, respectively. The first probe and the second probeinclude free ends 1107-1110. As shown in FIG. 11, the first and secondtarget sequences are not directly adjacent in the analyte. Afterhybridization, unbound first and second probes are washed away. Then, athird oligonucleotide 1106 hybridizes to the first and the second probesat 1108 and 1109, respectively. After hybridization, the first probe isextended 1112 and a ligation product is created that includes the firstprobe sequence and the second probe sequence. Alternatively, instead ofextending the first probe, the third oligonucleotide is used to “bind”the first probe and the second probe together. In such cases, the firstprobe and the second probe bound together by the third oligonucleotidecan be referred to as a ligation product. The ligation product then iscontacted with a substrate 1111, and the ligation product is bound to acapture probe 1113 of the substrate 1111 on the array at distinctspatial positions. In some embodiments, the biological sample iscontacted with the substrate 1111 prior to being contacted with thefirst probe and the second probe.

In some embodiments, the first oligonucleotide (e.g., the first probe)and the second oligonucleotide (e.g., the second probe) hybridize to ananalyte at a first temperature. In some embodiments, the firsttemperature ranges from about 50° C. to about 75° C., from about 55° C.to about 70° C., or from about 60° C. to about 65° C. In someembodiments, the first temperature is about 55° C., about 56° C., about57° C., about 58° C., about 59° C., about 60° C., about 61° C., about62° C., about 63° C., about 64° C., about 65° C., about 66° C., about67° C., about 68° C., about 69° C., or about 70° C.

In some embodiments, after the step of hybridizing the firstoligonucleotide (e.g., the first probe) and the second oligonucleotide(e.g., the second probe) to the analyte, a wash step is performed toremove unbound oligonucleotides (e.g., probes). The wash step can beperformed using any of the wash methods and solutions described herein.

In some embodiments, after the step of hybridizing the firstoligonucleotide (e.g., the first probe) and the second oligonucleotide(e.g., the second probe) to the analyte, a third oligonucleotide (e.g.,a splint oligonucleotide) is added to the analyte. In some embodiments,the third oligonucleotide is an oligonucleotide. In some embodiments,the third oligonucleotide is a DNA oligonucleotide.

In some embodiments, the third oligonucleotide includes a sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%complementary to a portion of the first probe oligonucleotide (e.g., aportion of the first probe that is not hybridized to the analyte (e.g.,an auxiliary sequence)). In some embodiments, the third oligonucleotideincludes a sequence that is 100% complementary to a portion of the firstoligonucleotide (e.g., the first probe). In some embodiments, the thirdoligonucleotide includes a sequence that is at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% complementary to a portion of thesecond probe oligonucleotide (e.g., a portion of the second probe thatis not hybridized to the analyte (e.g., an auxiliary sequence)). In someembodiments, the third oligonucleotide includes a sequence that is 100%complementary to a portion of the second oligonucleotide (e.g., thesecond probe). In some embodiments, the third oligonucleotide hybridizesto the first oligonucleotide (e.g., the first probe) at thecomplementary portion. In some embodiments, the third oligonucleotidehybridizes to the second oligonucleotide (e.g., the second probe) at thecomplementary portion.

In some embodiments, the third oligonucleotide hybridizes to the firstoligonucleotide (e.g., the first probe) and to the secondoligonucleotide (e.g., the second probe) at a second temperature. Insome embodiments, the second temperature is lower than the firsttemperature at which the first and second oligonucleotides (e.g., thefirst and second probes) bind the analyte. In some embodiments, thesecond temperature ranges from about 15° C. to about 35° C., from about20° C. to about 30° C., or from about 25° C. to about 30° C. In someembodiments, the first temperature is about 15° C., about 16° C., about17° C., about 18° C., about 19° C., about 20° C., about 21° C., about22° C., about 23° C., about 24° C., about 25° C., about 26° C., about27° C., about 28° C., about 29° C., about 30° C., about 31° C., about32° C., about 33° C., about 34° C., or about 35° C. Methods including athird, or splint, oligonucleotide have been described in U.S. PatentPub. No. 2019/0055594A1, which is herein incorporated by reference inits entirety.

In some embodiments, after the step of hybridizing the thirdoligonucleotide to the analyte, a wash step is performed to removeunbound third oligonucleotides. The wash step can be performed using anyof the wash methods and solutions described herein. In some embodiments,after the washing step, the first and second oligonucleotides (e.g., thefirst and second probes) are bound to (e.g., hybridized to) the analyte,and the third oligonucleotide is bound to (e.g., hybridized to) thefirst and second oligonucleotides (e.g., at portions of the first andsecond probes that are not bound to the analyte).

In some embodiments, the first oligonucleotide (e.g., the first probe),the second oligonucleotide (e.g., the second probe), and the thirdoligonucleotide are added to the biological sample at the same time.Then, in some embodiments, the temperature is adjusted to the firsttemperature to allow the first oligonucleotide (e.g., the first probe)and the second oligonucleotide (e.g., the second probe) to hybridize tothe analyte in the biological sample. Next, the temperature is adjustedto the second temperature to allow the third oligonucleotide tohybridize to the first oligonucleotide and the second oligonucleotide.

In some embodiments where a third oligonucleotide hybridizes to a firstprobe and a second probe that are hybridized to targets sequences thatare not directly adjacent in the analyte, the third oligonucleotide isextended to fill the gap between the first probe and the second probe.In some instances, a polymerase (e.g., a DNA polymerase) can extend oneof the probes (e.g., the first probe) prior to ligation. For example, asshown in FIG. 11, the first probe 1102 is extended 1112 to fill the gap1114 between the first probe 1102 and the second probe 1103.

In some embodiments, a ligation step is performed. Ligation can beperformed using any of the methods described herein. In someembodiments, the step includes ligation of the first oligonucleotide(e.g., the first probe) and the second oligonucleotide (e.g., the secondprobe), forming a ligation product. In some embodiments, the thirdoligonucleotide serves as an oligonucleotide splint to facilitateligation of the first oligonucleotide (e.g., the first probe) and thesecond oligonucleotide (e.g., the second probe). In some embodiments,ligation is chemical ligation. In some embodiments, ligation isenzymatic ligation. In some embodiments, the ligase is a T4 RNA ligase(Rnl2), a splintR ligase, a single stranded DNA ligase, or a T4 DNAligase.

(ii) Hybridization Buffer

In some embodiments, a first probe and a second probe are hybridized tothe analyte in a hybridization buffer. In some instances, thehybridization buffer contains formamide. In other instances thehybridization buffer is formamide free. Formamide is not human friendlyand it is a known health hazard. Chemically, it can oxidize over time,thereby impacting reagent shelf life and, most importantly, reagentefficacy. As such, the methods described herein can includeformamide-free buffers, including formamide-free hybridization buffer.

In some embodiments, the formamide-free hybridization buffer is asaline-sodium citrate (SSC) hybridization buffer. In some embodiment,the SSC is present in the SSC hybridization buffer from about 1×SSC toabout 6×SSC (e.g., about 1×SSC to about 5×SSC, about 1×SSC to about4×SSC, about 1×SSC to about 3×SSC, about 1×SSC to about 2×SSC, about2×SSC to about 6×SSC, about 2×SSC to about 5×SSC, about 2×SSC to about4×SSC, about 2×SSC to about 3×SSC, about 3×SSC to about 5×SSC, about3×SSC to about 4×SSC, about 4×SSC to about 6×SSC, about 4×SSC to about6×SSC, about 4×SSC to about 5×SSC, or about 5×SSC to about 6×SSC). Insome embodiments, the SSC is present in the SSC hybridization bufferfrom about 2×SSC to about 4×SSC. In some embodiments, SSPE hybridizationbuffer can be used.

In some embodiments, the SSC hybridization buffer comprises a solvent.In some embodiments, the solvent comprises ethylene carbonate instead offormamide (2020, Kalinka et al., Scientia Agricola 78(4):e20190315). Insome embodiments, ethylene carbonate is present in the SSC hybridizationbuffer from about 10% (w/v) to about 25% (w/v) (e.g., about 10% (w/v) toabout 20% (w/v), about 10% (w/v) to about 15% (w/v), about 15% (w/v) toabout 25% (w/v), about 15% (w/v) to about 20% (w/v), or about 20% (w/v)to about 25% (w/v)). In some embodiments, ethylene carbonate is presentin the SSC hybridization buffer from about 15% (w/v) to about 20% (w/v).In some embodiments, ethylene carbonate is present in the SSChybridization buffer at about 10% (w/v), about 11% (w/v), about 12%(w/v), about 13% (w/v), about 14% (w/v), about 15% (w/v), about 16%(w/v), about 17% (w/v), about 18% (w/v), about 19% (w/v), about 20%(w/v), about 21% (w/v), about 22% (w/v), about 23% (w/v), about 24%(w/v), or about 25% (w/v). In some embodiments, ethylene carbonate ispresent in the SSC hybridization buffer at about 13% (w/v).

In some embodiments, the SSC hybridization buffer is at a temperaturefrom about 40° C. to about 60° C. (e.g., about 40° C. to about 55° C.,about 40° C. to about 50° C., about 40° C. to about 45° C., about 45° C.to about 60° C., about 45° C. to about 55° C., about 45° C. to about 50°C., about 50° C. to about 60° C., about 50° C. to about 55° C., or about55° C. to about 60° C.). In some embodiments, the SSC hybridizationbuffer is at temperature from about 45° C. to about 55° C., or any ofthe subranges described herein. In some embodiments, the SSChybridization buffer is at a temperature of about 40° C., about 41° C.,about 42° C., about 43° C., about 44° C., about 45° C., about 46° C.,about 47° C., about 48° C., about 49° C., about 50° C., about 51° C.,about 52° C., about 53° C., about 54° C., about 55° C., about 56° C.,about 57° C., about 58° C., about 59° C., or about 60° C. In someembodiments, the SSC hybridization buffer is at a temperature of about50° C.

In some embodiments, the SSC hybridization buffer further comprises oneor more of a carrier, a crowder, or an additive. Non-limiting examplesof a carrier that can be included in the hybridization buffer include:yeast tRNA, salmon sperm DNA, lambda phage DNA, glycogen, andcholesterol. Non-limiting examples of a molecular crowder that can beincluded in the hybridization buffer include: Ficoll, dextran,Denhardt's solution, and PEG. Non-limiting examples of additives thatcan be included in the hybridization buffer include: binding blockers,RNase inhibitors, Tm adjustors and adjuvants for relaxing secondarynucleic acid structures (e.g., betaine, TMAC, and DMSO). Further, ahybridization buffer can include detergents such as SDS, Tween, Triton-X100, and sarkosyl (e.g., N-Lauroylsarcosine sodium salt). A skilledartisan would understand that a buffer for hybridization of nucleicacids could include many different compounds that could enhance thehybridization reaction.

(f) Washing

In some embodiments, the methods disclosed herein also include a washstep. The wash step removes any unbound probes. Wash steps could beperformed between any of the steps in the methods disclosed herein. Forexample, a wash step can be performed after adding probes to thebiological sample. As such, free/unbound probes are washed away, leavingonly probes that have hybridized to an analyte. In some instances,multiple (i.e., at least 2, 3, 4, 5, or more) wash steps occur betweenthe methods disclosed herein. Wash steps can be performed at times(e.g., 1, 2, 3, 4, or 5 minutes) and temperatures (e.g., roomtemperature; 4° C. known in the art and determined by a person of skillin the art.

In some instances, wash steps are performed using a wash buffer. In someinstances, the wash buffer includes SSC (e.g., 1×SSC). In someinstances, the wash buffer includes PBS (e.g., 1×PBS). In someinstances, the wash buffer includes PBST (e.g., 1×PBST). In someinstances, the wash buffer can also include formamide or be formamidefree.

Additional embodiments regarding wash steps are provided herein.

(i) Formamide Free Wash Buffer

In some embodiments, after ligating a first probe and a second probe,the one or more unhybridized first probes, one or more unhybridizedsecond probes, or both, are removed from the array. In some embodiments,after ligating a first probe, one or more spanning probes, and a secondprobe, the one or more unhybridized first, second, and/or spanningprobes, are removed from the array. In some embodiments, after ligatinga first probe, a second probe, and a third oligonucleotide, the one ormore unhybridized first probes, one or more unhybridized second probes,or one or more third oligonucleotides, or all the above, are removedfrom the array.

In some embodiments, a pre-hybridization buffer is used to wash thesample. In some embodiments, a phosphate buffer is used. In someembodiments, multiple wash steps are performed to remove unboundoligonucleotides.

In some embodiments, removing includes washing the one or moreunhybridized probes (e.g., a first probe, a second probe, a spanningprobe, additional spanning probes, and a third oligonucleotide) from thearray in a formamide-free wash buffer.

In some embodiments, the formamide-free wash buffer is an SSC washbuffer. In some embodiments, SSC is present in the SSC wash buffer fromabout 0.01×SSC to about 1×SSC (e.g., about 0.01×SSC to about 0.5×SSC,0.01×SSC to about 0.1×SSC, about 0.01×SSC to about 0.05×SSC, about0.05×SSC to about 1×SSC, about 0.05×SSC to about 0.5×SSC, about 0.05×SSCto about 0.1×SSC, about 0.1×SSC to about 1×SSC, about 0.1×SSC to about0.5×SSC, or about 0.5×SSC to about 1×SSC). In some embodiments, SSC ispresent in the SSC wash buffer at about 0.01×SSC, about 0.02×SSC, about0.03×SSC, about 0.04×SSC, about 0.05×SSC, about 0.06×SSC, about0.07×SSC, about 0.08×SSC, about 0.09×SSC, about 0.1×SSC, about 0.2×SSC,about 0.3×SSC, about 0.4×SSC, about 0.5×SSC, about 0.6×SSC, about0.7×SSC, about 0.8×SSC, about 0.9×SSC, or about 0.1×SSC. In someembodiments, SSC is present in the SSC wash buffer at about 0.1×SSC.

In some embodiments, the SSC wash buffer comprises a detergent. In someembodiments, the detergent comprises sodium dodecyl sulfate (SDS). Insome embodiments, SDS is present in the SSC wash buffer from about 0.01%(v/v) to about 0.5% (v/v) (e.g., about 0.01% (v/v) to about 0.4% (v/v),about 0.01% (v/v) to about 0.3% (v/v), about 0.01% (v/v) to about 0.2%(v/v), about 0.01% (v/v) to about 0.1% (v/v), about 0.05% (v/v) to about0.5% (v/v), about 0.05% (v/v) to about 0.4% (v/v), about 0.05% (v/v) toabout 0.3% (v/v), about 0.05% (v/v) to about 0.2% (v/v), about 0.05%(v/v) to about 0.1% (v/v), about 0.1% (v/v) to about 0.5% (v/v), about0.1% (v/v) to about 0.4% (v/v), about 0.1% (v/v) to about 0.3% (v/v),about 0.1% (v/v) to about 0.2% (v/v), about 0.2% (v/v) to about 0.5%(v/v), about 0.2% (v/v) to about 0.4% (v/v), about 0.2% (v/v) to about0.3% (v/v), about 0.3% (v/v) to about 0.5% (v/v), about 0.3% (v/v) toabout 0.4% (v/v), or about 0.4% (v/v) to about 0.5% (v/v)). In someembodiments, the SDS is present the SSC wash buffer at about 0.01%(v/v), about 0.02% (v/v), about 0.03% (v/v), about 0.04% (v/v), about0.05% (v/v), about 0.06% (v/v), about 0.07% (v/v), about 0.08% (v/v),about 0.09% (v/v), about 0.10% (v/v), about 0.2% (v/v), about 0.3%(v/v), about 0.4% (v/v), or about 0.5% (v/v), In some embodiments, theSDS is present in the SSC wash buffer at about 0.1% (v/v). In someembodiments, sarkosyl may be present in the SSC wash buffer.

In some embodiments, the SSC wash buffer comprises a solvent. In someembodiments, the solvent comprises ethylene carbonate. In someembodiments, ethylene carbonate is present in the SSC wash buffer fromabout 10% (w/v) to about 25% (w/v), or any of the subranges describedherein. In some embodiments, ethylene carbonate is present in the SSCwash buffer from about 15% (w/v) to about 20% (w/v). In someembodiments, ethylene carbonate is present in the SSC wash buffer atabout 16% (w/v).

In some embodiments, the SSC wash buffer is at a temperature from about50° C. to about 70° C. (e.g., about 50° C. to about 65° C., about 50° C.to about 60° C., about 50° C. to about 55° C., about 55° C. to about 70°C., about 55° C. to about 65° C., about 55° C. to about 60° C., about60° C. to about 70° C., about 60° C. to about 65° C., or about 65° C. toabout 70° C.). In some embodiments, the SSC wash buffer is at atemperature from about 55° C. to about 65° C. In some embodiments, theSSC wash buffer is at a temperature about 50° C., about 51° C., about52° C., about 53° C., about 54° C., about 55° C., about 56° C., about57° C., about 58° C., about 59° C., about 60° C., about 61° C., about62° C., about 63° C., about 64° C., about 65° C., about 66° C., about67° C., about 68° C., about 69° C., or about 70° C. In some embodiments,the SSC wash buffer is at a temperature of about 60° C.

In some embodiments, the method includes releasing the ligation product,where releasing is performed after the array is washed to remove the oneor more unhybridized first and second probes.

(g) Ligation

In some embodiments, after hybridization of the probe oligonucleotides(e.g., a first probe, a second probe, a spanning probe, additionalspanning probes, and/or a third oligonucleotide) to the analyte, theprobe (e.g., a first probe, a second probe, a spanning probe, additionalspanning probes, and/or a third oligonucleotide) can be ligatedtogether, creating a single ligation product that includes one or moresequences that are complementary to the analyte. Ligation can beperformed enzymatically or chemically, as described herein.

In some instances, the ligation is an enzymatic ligation reaction, usinga ligase (e.g., T4 RNA ligase (Rnl2), a SplintR ligase, a singlestranded DNA ligase, or a T4 DNA ligase). See, e.g., Zhang et al.; RNABiol. 2017; 14(1): 36-44, which is incorporated by reference in itsentirety, for a description of KOD ligase. Following the enzymaticligation reaction, the probes (e.g., a first probe, a second probe, aspanning probe, additional spanning probes, and/or a thirdoligonucleotide) may be considered ligated.

In some embodiments, a polymerase catalyzes synthesis of a complementarystrand of the ligation product, creating a double-stranded ligationproduct. In some instances, the polymerase is DNA polymerase. In someembodiments, the polymerase has 5′ to 3′ polymerase activity. In someembodiments, the polymerase has 3′ to 5′ exonuclease activity forproofreading. In some embodiments, the polymerase has 5′ to 3′polymerase activity and 3′ to 5′ exonuclease activity for proofreading.

In some embodiments, the probe (e.g., a first probe, a second probe, aspanning probe, additional spanning probes, and/or a thirdoligonucleotide) may each comprise a reactive moiety such that, uponhybridization to the target and exposure to appropriate ligationconditions, the probe oligonucleotides may ligate to one another. Insome embodiments, probe oligonucleotides that include a reactive moietyare ligated chemically. For example, a first probe capable ofhybridizing to a first target region (e.g., a first target sequence or afirst portion) of a nucleic acid molecule may comprise a first reactivemoiety, and a second probe oligonucleotide capable of hybridizing to asecond target region (e.g., a second target sequence or a secondportion) of the nucleic acid molecule may comprise a second reactivemoiety. When the first and second probes are hybridized to the first andsecond target regions (e.g., first and second target sequences) of thenucleic acid molecule, the first and second reactive moieties may beadjacent to one another. A reactive moiety of a probe may be selectedfrom the non-limiting group consisting of azides, alkynes, nitrones(e.g., 1,3-nitrones), strained alkenes (e.g., trans-cycloalkenes such ascyclooctenes or oxanorbornadiene), tetrazines, tetrazoles, iodides,thioates (e.g., phorphorothioate), acids, amines, and phosphates. Forexample, the first reactive moiety of a first probe may comprise anazide moiety, and a second reactive moiety of a second probe maycomprise an alkyne moiety. The first and second reactive moieties mayreact to form a linking moiety. A reaction between the first and secondreactive moieties may be, for example, a cycloaddition reaction such asa strain-promoted azide-alkyne cycloaddition, a copper-catalyzedazide-alkyne cycloaddition, a strain-promoted alkyne-nitronecycloaddition, a Diels-Alder reaction, a [3+2] cycloaddition, a [4+2]cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; anucleophilic substation reaction; or another reaction. In some cases,reaction between the first and second reactive moieties may yield atriazole moiety or an isoxazoline moiety. A reaction between the firstand second reactive moieties may involve subjecting the reactivemoieties to suitable conditions such as a suitable temperature, pH, orpressure and providing one or more reagents or catalysts for thereaction. For example, a reaction between the first and second reactivemoieties may be catalyzed by a copper catalyst, a ruthenium catalyst, ora strained species such as a difluorooctyne, dibenzylcyclooctyne, orbiarylazacyclooctynone. Reaction between a first reactive moiety of afirst probe hybridized to a first target region (e.g., a first targetsequence or first portion) of the nucleic acid molecule and a secondreactive moiety of a third probe oligonucleotide hybridized to a secondtarget region (e.g., a first target sequence or a first portion) of thenucleic acid molecule may link the first probe and the second probe toprovide a ligated probe. Upon linking, the first and second probe may beconsidered ligated. Accordingly, reaction of the first and secondreactive moieties may comprise a chemical ligation reaction such as acopper-catalyzed 5′ azide to 3′ alkyne “click” chemistry reaction toform a triazole linkage between two probe oligonucleotides. In othernon-limiting examples, an iodide moiety may be chemically ligated to aphosphorothioate moiety to form a phosphorothioate bond, an acid may beligated to an amine to form an amide bond, and/or a phosphate and aminemay be ligated to form a phosphoramidate bond.

FIGS. 12A-12E illustrates examples of representative reactions. FIG. 12Ashows a chemical ligation reaction of an alkyne moiety 1202 and an azidemoiety 1204 reacting under copper-mediated cycloaddition to form atriazole linkage 1206. FIG. 12B shows a chemical ligation reaction of aphosphorothioate group 1208 with an iodide group 1210 to form aphosphorothioate linkage 1212. FIG. 12C shows a chemical ligationreaction of an acid 1214 and amine 1216 to form an amide linkage 1218.FIG. 12D shows a chemical ligation reaction of a phosphate moiety 1220and an amine moiety 1222 to form a phosphoramidate linkage 1224. FIG.12E shows a conjugation reaction of two species 1226 and 1228.

In some instances, ligation is performed in a ligation buffer. Ininstances where probe ligation is performed on diribo-containing probes,the ligation buffer can include T4 RNA Ligase Buffer 2, enzyme (e.g.,RNL2 ligase), and nuclease free water. In instances where probe ligationis performed on DNA probes, the ligation buffer can include Tris-HClpH7.5, MnCl2, ATP, DTT, surrogate fluid (e.g., glycerol), enzyme (e.g.,SplintR ligase), and nuclease-free water.

In some embodiments, the ligation buffer includes additional reagents.In some instances, the ligation buffer includes adenosine triphosphate(ATP) is added during the ligation reaction. DNA ligase-catalyzedsealing of nicked DNA substrates is first activated through ATPhydrolysis, resulting in covalent addition of an AMP group to theenzyme. After binding to a nicked site in a DNA duplex, the ligasetransfers the AMP to the phosphorylated 5′-end at the nick, forming a5′-5′ pyrophosphate bond. Finally, the ligase catalyzes an attack onthis pyrophosphate bond by the OH group at the 3′-end of the nick,thereby sealing it, whereafter ligase and AMP are released. If theligase detaches from the substrate before the 3′ attack, e.g. because ofpremature AMP reloading of the enzyme, then the 5′ AMP is left at the5′-end, blocking further ligation attempts. In some instances, ATP isadded at a concentration of about 10 μM, about 100 μM, about 1000 μM, orabout 10000 μM during the ligation reaction.

In some embodiments, cofactors that aid in joining of the probeoligonucleotides are added during the ligation process. In someinstances, the cofactors include magnesium ions (Mg²⁺). In someinstances, the cofactors include manganese ions (Mn²⁺). In someinstances, Mg²⁺ is added in the form of MgCl₂. In some instances, Mn²⁺is added in the form of MnCl₂. In some instances, the concentration ofMgCl₂ is at about 1 mM, at about 10 mM, at about 100 mM, or at about1000 mM. In some instances, the concentration of MnCl₂ is at about 1 mM,at about 10 mM, at about 100 mM, or at about 1000 mM.

In some embodiments, the ligation product includes a capture probecapture domain, which can bind to a capture probe (e.g., a capture probeimmobilized, directly or indirectly, on a substrate). In someembodiments, methods provided herein include contacting a biologicalsample with a substrate, wherein the capture probe is affixed to thesubstrate (e.g., immobilized to the substrate, directly or indirectly).In some embodiments, the capture probe capture domain of the ligatedprobe specifically binds to the capture domain.

After ligation, in some instances, the biological sample is washed witha post-ligation wash buffer. In some instances, the post-ligation washbuffer includes one or more of SSC (e.g., 1×SSC), ethylene carbonate orformamide, and nuclease free water. In some instances, the biologicalsample is washed at this stage at about 50° C. to about 70° C. In someinstances, the biological sample is washed at about 60° C.

(i) Ligation Including Pre-Adenylated 5′ Phosphate on Second Probe

Provided herein are methods for determining a location of a targetnucleic acid in a biological sample that include: (a) contacting thebiological sample with a substrate comprising a plurality of captureprobes, where a capture probe of the plurality of capture probescomprises a capture domain and a spatial barcode; (b) hybridizing atarget nucleic acid in the biological sample with a first probe and asecond probe, where the first probe comprises, from 3′ to 5′, a sequencesubstantially complementary to the capture domain and a sequence that issubstantially complementary to a first sequence in the target nucleicacid and has a pre-adenylated phosphate group at its 5′ end; the secondprobe comprises a sequence substantially complementary to a secondsequence in the target nucleic acid; (c) generating a ligation productby ligating a 3′ end of the second probe to the 5′ end of the firstprobe using a ligase that does not require adenosine triphosphate forligase activity; (d) releasing the ligation product from the targetnucleic acid and binding the capture domain of the ligation productspecifically to the capture domain of capture probe; and (e) determining(i) all or a part of a sequence corresponding to the ligation product,or a complement thereof, and (ii) all or a part of a sequencecorresponding to the spatial barcode, or a complement thereof, and usingthe determined sequences of (i) and (ii) to identify the location of thetarget nucleic acid in the biological sample

In some instances, the ligase that does not require adenosinetriphosphate for ligase activity (e.g., thermostable 5′ AppDNA/RNALigase, truncated T4 RNA Ligase 2 (trRnl2), truncated T4 RNA Ligase 2K227Q, truncated T4 RNA Ligase 2 KQ, Chlorella Virus PBCV-1 DNA Ligase,and combinations thereof). See, e.g., Nichols et al., “RNA Ligases,”Curr. Protocol. Molec. Biol. 84(1):3.15.1-0.4 (2008); Viollet et al.,“T4 RNA Ligase 2 Truncated Active Site Mutants: Improved Tools for RNAAnalysis,” BMC Biotechnol. 11: 72 (2011); and Ho et al., “BacteriophageT4 RNA Ligase 2 (gp24.1) Exemplifies a Family of RNA Ligases Found inAll Phylogenetic Domains,” PNAS 99(20):12709-14 (2002), which are herebyincorporated by reference in their entirety for a description of T4 RNALigases and truncated T4 RNA Ligases. Thermostable 5′ AppDNA/RNA Ligaseis an enzyme belonging to the Ligase family that catalyzes the ligationof the 3′ end of ssRNA or ssDNA to a 5′-adenylated ssDNA or5′-adenylated ssRNA. Truncated T4 RNA Ligase 2 is an enzyme belonging tothe Ligase family that catalyzes the ligation of dsRNA nicks and ssRNAto ssRNA. It can also ligate the 3′ end of RNA or DNA to a 5′-pDNA whenannealed to an RNA complement, and the 3′ end of RNA to a 5′-pRNA whenannealed to a DNA complement, with reduced efficiency. Truncated T4 RNALigase 2 K227Q is an enzyme belonging to the Ligase family thatcatalyzes the ligation of the 3′ end of ssRNA to 5′ adenylated ssDNA and5′ adenylated ssRNA. It has a reduction of side products as compared totruncated T4 RNA Ligase 2. Truncated T4 RNA Ligase 2 KQ is an enzymebelonging to the Ligase family that catalyzes the ligation of the 3′ endof ssRNA to 5′ adenylated ssDNA and 5′ adenylated ssRNA. It is apreferred choice for ligation of ssRNA to preadenylated adapters and hasa reduction of side products as compared to truncated T4 RNA Ligase 2.

In some embodiments, the T4 RNA Ligase comprises a K227Q mutation. SeeViollet et al., “T4 RNA Ligase 2 Truncated Active Site Mutants: ImprovedTools for RNA Analysis,” BMC Biotechnol. 11, which is herebyincorporated by reference in its entirety.

In some instances, cofactors that aid in ligation of the first andsecond probe are added during ligation. In some instances, the cofactorsinclude magnesium ions (Mg²⁺). In some instances, the cofactors includemanganese ions (Mn²⁺). In some instances, Mg²⁺ is added in the form ofMgCl₂. In some instances, Mn²⁺ is added in the form of MnCl₂. In someinstances, the concentration of MgCl₂ is at about 1 mM to about 10 mM.In some instances, the concentration of MnCl₂ is at about 1 mM to about10 mM.

In some instances, the ligation occurs at a pH in the range of about 6.5to about 9.0, about 6.5 to about 8.0, or about 7.5 to about 8.0.

In some embodiments, the ligation buffer includes an enzyme storagebuffer. In some embodiments, the enzymes storage buffer includesglycerol. In some embodiments, the ligation buffer is supplemented withglycerol. In some embodiments, the glycerol is present in the ligationbuffer at a total volume of 15% v/v.

(h) Permeabilization and Releasing the Ligation Product

In some embodiments, the methods provided herein include apermeabilizing step. In some embodiments, permeabilization occurs usinga protease. In some embodiments, the protease is an endopeptidase.Endopeptidases that can be used include but are not limited to trypsin,chymotrypsin, elastase, thermolysin, pepsin, clostripan, glutamylendopeptidase (GluC), ArgC, peptidyl-asp endopeptidase (ApsN),endopeptidase LysC and endopeptidase LysN. In some embodiments, theendopeptidase is pepsin. In some embodiments, after creating a ligationproduct (e.g., by ligating a first probe and a second probe that arehybridized to adjacent sequences in the analyte), the biological sampleis permeabilized. In some embodiments, the biological sample ispermeabilized contemporaneously with or prior to contacting thebiological sample with a first probe and a second probe, hybridizing thefirst probe and the second probe to the analyte, generating a ligationproduct by ligating the first probe and the second probe, and releasingthe ligated product from the analyte.

In some embodiments, methods provided herein include permeabilization ofthe biological sample such that the capture probe can more easily bindto the captured ligated probe (i.e., compared to no permeabilization).In some embodiments, reverse transcription (RT) reagents can be added topermeabilized biological samples. Incubation with the RT reagents canproduce spatially-barcoded full-length cDNA from the captured analytes(e.g., polyadenylated mRNA). Second strand reagents (e.g., second strandprimers, enzymes) can be added to the biological sample on the slide toinitiate second strand synthesis.

In some instances, the permeabilization step includes application of apermeabilization buffer to the biological sample. In some instances, thepermeabilization buffer includes a buffer (e.g., Tris pH 7.5), MgCl2,sarkosyl detergent (e.g., sodium lauroyl sarcosinate), enzyme (e.g.,proteinase K, and nuclease free water. In some instances, thepermeabilization step is performed at 37° C. In some instances, thepermeabilization step is performed for about 20 minutes to 2 hours(e.g., about 20 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 1 hour, about 1.5 hours, or about 2 hours). In someinstances, the releasing step is performed for about 40 minutes.

In some embodiments, after generating a ligation product, the ligationproduct is released from the analyte. In some embodiments, a ligationproduct is released from the analyte using an endoribonuclease. In someembodiments, the endoribonuclease is RNase H, RNase A, RNase C, or RNaseI. In some embodiments, the endoribonuclease is RNase H. RNase H is anendoribonuclease that specifically hydrolyzes the phosphodiester bondsof RNA, when hybridized to DNA. RNase H is part of a conserved family ofribonucleases which are present in many different organisms. There aretwo primary classes of RNase H: RNase H1 and RNase H2. Retroviral RNaseH enzymes are similar to the prokaryotic RNase H1. All of these enzymesshare the characteristic that they are able to cleave the RNA componentof an RNA:DNA heteroduplex. In some embodiments, the RNase H is RNaseH1, RNase H2, or RNase H1, or RNase H2. In some embodiments, the RNase Hincludes but is not limited to RNase HII from Pyrococcus furiosus, RNaseHII from Pyrococcus horikoshi, RNase HI from Thermococcus litoralis,RNase HI from Thermus thermophilus, RNAse HI from E. coli, or RNase HIIfrom E. coli.

In some instances, the releasing step is performed using a releasingbuffer. In some instances, the release buffer includes one or more of abuffer (e.g., Tris pH 7.5), enzyme (e.g., RNAse H) and nuclease-freewater. In some instances, the releasing step is performed at 37° C. Insome instances, the releasing step is performed for about 20 minutes to2 hours (e.g., about 20 minutes, about 30 minutes, about 40 minutes,about 50 minutes, about 1 hour, about 1.5 hours, or about 2 hours). Insome instances, the releasing step is performed for about 30 minutes.

In some instances, the releasing step occurs before the permeabilizationstep. In some instances, the releasing step occurs after thepermeabilization step. In some instances, the releasing step occurs atthe same time as the permeabilization step (e.g., in the same buffer).

(i) Blocking Probes

In some embodiments, a capture probe capture domain is blocked prior toadding a second probe oligonucleotide to a biological sample. Thisprevents the capture probe capture domain from prematurely hybridizingto the capture domain.

In some embodiments, a blocking probe is used to block or modify thefree 3′ end of the capture probe capture domain. In some embodiments, ablocking probe can be hybridized to the capture probe capture domain ofthe second probe to mask the free 3′ end of the capture probe capturedomain. In some embodiments, a blocking probe can be a hairpin probe orpartially double stranded probe. In some embodiments, the free 3′ end ofthe capture probe capture domain of the second probe can be blocked bychemical modification, e.g., addition of an azidomethyl group as achemically reversible capping moiety such that the capture probes do notinclude a free 3′ end. Blocking or modifying the capture probe capturedomain, particularly at the free 3′ end of the capture probe capturedomain, prior to contacting second probe with the substrate, preventshybridization of the second probe to the capture domain (e.g., preventsthe capture of a poly(A) of a capture probe capture domain to a poly(T)capture domain). In some embodiments, a blocking probe can be referredto as a capture probe capture domain blocking moiety.

In some embodiments, the blocking probes can be reversibly removed. Forexample, blocking probes can be applied to block the free 3′ end ofeither or both the capture probe capture domain and/or the captureprobes. Blocking interaction between the capture probe capture domainand the capture probe on the substrate can reduce non-specific captureto the capture probes. After the second probe hybridizes to the analyteand is ligated to a first probe, one or more spanning probes, or a thirdoligonucleotide, the blocking probes can be removed from the 3′ end ofthe capture probe capture domain and/or the capture probe, and theligation product can migrate to and become bound by the capture probeson the substrate. In some embodiments, the removal includes denaturingthe blocking probe from capture probe capture domain and/or captureprobe. In some embodiments, the removal includes removing a chemicallyreversible capping moiety. In some embodiments, the removal includesdigesting the blocking probe with an RNase (e.g., RNase H).

In some embodiments, the blocking probes are oligo (dT) blocking probes.In some embodiments, the oligo (dT) blocking probes can have a length of15-30 nucleotides. In some embodiments, the oligo (dT) blocking probescan have a length of 10-50 nucleotides, e.g., 10-50, 10-45, 10-40,10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30,15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45,25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40,40-50, 40-45, or 45-50 nucleotides. In some embodiments, the analytecapture agents can be blocked at different temperatures (e.g., 4° C. and37° C.).

(j) Biological Samples

Methods disclosed herein can be performed on any type of sample. In someembodiments, the sample is a fresh tissue. In some embodiments, thesample is a frozen sample. In some embodiments, the sample waspreviously frozen. In some embodiments, the sample is a formalin-fixed,paraffin embedded (FFPE) sample.

Subjects from which biological samples can be obtained can be healthy orasymptomatic individuals, individuals that have or are suspected ofhaving a disease (e.g., cancer) or a pre-disposition to a disease,and/or individuals that are in need of therapy or suspected of needingtherapy. In some instances, the biological sample can include one ormore diseased cells. A diseased cell can have altered metabolicproperties, gene expression, protein expression, and/or morphologicfeatures. Examples of diseases include inflammatory disorders, metabolicdisorders, nervous system disorders, and cancer. In some instances, thebiological sample includes cancer or tumor cells. Cancer cells can bederived from solid tumors, hematological malignancies, cell lines, orobtained as circulating tumor cells. In some instances, the biologicalsample is a heterogenous sample. In some instances, the biologicalsample is a heterogenous sample that includes tumor or cancer cellsand/or stromal cells,

In some instances, the cancer is breast cancer. In some instances, thebreast cancer is triple positive breast cancer (TPBC). In someinstances, the breast cancer is triple negative breast cancer (TNBC).

In some instances, the cancer is colorectal cancer. In some instances,the cancer is ovarian cancer. In certain embodiments, the cancer issquamous cell cancer, small-cell lung cancer, non-small cell lungcancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin's lymphoma,pancreatic cancer, glioblastoma, glioma, cervical cancer, ovariancancer, liver cancer, bladder cancer, breast cancer, colon cancer,colorectal cancer, endometrial carcinoma, myeloma, salivary glandcarcinoma, kidney cancer, basal cell carcinoma, melanoma, prostatecancer, vulval cancer, thyroid cancer, testicular cancer, esophagealcancer, or a type of head or neck cancer. In certain embodiments, thecancer treated is desmoplastic melanoma, inflammatory breast cancer,thymoma, rectal cancer, anal cancer, or surgically treatable ornon-surgically treatable brain stem glioma. In some embodiments, thesubject is a human.

FFPE samples generally are heavily cross-linked and fragmented, andtherefore this type of sample allows for limited RNA recovery usingconventional detection techniques. In certain embodiments, methods oftargeted RNA capture provided herein are less affected by RNAdegradation associated with FFPE fixation than other methods (e.g.,methods that take advantage of oligo-dT capture and reversetranscription of mRNA). In certain embodiments, methods provided hereinenable sensitive measurement of specific genes of interest thatotherwise might be missed with a whole transcriptomic approach.

In some instances, FFPE samples are stained (e.g., using H&E). Themethods disclosed herein are compatible with H&E will allow formorphological context overlaid with transcriptomic analysis. However,depending on the need some samples may be stained with only a nuclearstain, such as staining a sample with only hematoxylin and not eosin,when location of a cell nucleus is needed.

In some embodiments, a biological sample (e.g. tissue section) can befixed with methanol, stained with hematoxylin and eosin, and imaged. Insome embodiments, fixing, staining, and imaging occurs before one ormore probes are hybridized to the sample. Some embodiments of any of theworkflows described herein can further include a destaining step (e.g.,a hematoxylin and eosin destaining step), after imaging of the sampleand prior to permeabilizing the sample. For example, destaining can beperformed by performing one or more (e.g., one, two, three, four, orfive) washing steps (e.g., one or more (e.g., one, two, three, four, orfive) washing steps performed using a buffer including HCl). The imagescan be used to map spatial gene expression patterns back to thebiological sample. A permeabilization enzyme can be used to permeabilizethe biological sample directly on the slide.

In some embodiments, the FFPE sample is deparaffinized, permeabilized,equilibrated, and blocked before target probe oligonucleotides areadded. In some embodiments, deparaffinization using xylenes. In someembodiments, deparaffinization includes multiple washes with xylenes. Insome embodiments, deparaffinization includes multiple washes withxylenes followed by removal of xylenes using multiple rounds of gradedalcohol followed by washing the sample with water. In some aspects, thewater is deionized water. In some embodiments, equilibrating andblocking includes incubating the sample in a pre-Hyb buffer. In someembodiments, the pre-Hyb buffer includes yeast tRNA. In someembodiments, permeabilizing a sample includes washing the sample with aphosphate buffer. In some embodiments, the buffer is PBS. In someembodiments, the buffer is PBST.

(k) Determining the Sequence of the Ligation Product

After a ligation product from the sample has hybridized or otherwisebeen associated with a capture probe according to any of the methodsdescribed above in connection with the general spatial cell-basedanalytical methodology, the barcoded constructs that result fromhybridization/association are analyzed.

In some embodiments, after contacting a biological sample with asubstrate that includes capture probes, a removal step can optionally beperformed to remove all or a portion of the biological sample from thesubstrate. In some embodiments, the removal step includes enzymaticand/or chemical degradation of cells of the biological sample. Forexample, the removal step can include treating the biological samplewith an enzyme (e.g., a proteinase, e.g., proteinase K) to remove atleast a portion of the biological sample from the substrate. In someembodiments, the removal step can include ablation of the tissue (e.g.,laser ablation).

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample), the method comprising: (a) optionally stainingand/or imaging a biological sample on a substrate; (b) permeabilizing(e.g., providing a solution comprising a permeabilization reagent to)the biological sample on the substrate; (c) contacting the biologicalsample with an array comprising a plurality of capture probes, wherein acapture probe of the plurality captures the biological analyte; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte; wherein the biological sample is fully orpartially removed from the substrate.

In some embodiments, a biological sample is not removed from thesubstrate. For example, the biological sample is not removed from thesubstrate prior to releasing a capture probe (e.g., a capture probebound to an analyte) from the substrate. In some embodiments, suchreleasing comprises cleavage of the capture probe from the substrate(e.g., via a cleavage domain). In some embodiments, such releasing doesnot comprise releasing the capture probe from the substrate (e.g., acopy of the capture probe bound to an analyte can be made and the copycan be released from the substrate, e.g., via denaturation). In someembodiments, the biological sample is not removed from the substrateprior to analysis of an analyte bound to a capture probe after it isreleased from the substrate. In some embodiments, the biological sampleremains on the substrate during removal of a capture probe from thesubstrate and/or analysis of an analyte bound to the capture probe afterit is released from the substrate. In some embodiments, the biologicalsample remains on the substrate during removal (e.g., via denaturation)of a copy of the capture probe (e.g., complement). In some embodiments,analysis of an analyte bound to capture probe from the substrate can beperformed without subjecting the biological sample to enzymatic and/orchemical degradation of the cells (e.g., permeabilized cells) orablation of the tissue (e.g., laser ablation).

In some embodiments, at least a portion of the biological sample is notremoved from the substrate. For example, a portion of the biologicalsample can remain on the substrate prior to releasing a capture probe(e.g., a capture prove bound to an analyte) from the substrate and/oranalyzing an analyte bound to a capture probe released from thesubstrate. In some embodiments, at least a portion of the biologicalsample is not subjected to enzymatic and/or chemical degradation of thecells (e.g., permeabilized cells) or ablation of the tissue (e.g., laserablation) prior to analysis of an analyte bound to a capture probe fromthe substrate.

In some embodiments, provided herein are methods for spatially detectingan analyte (e.g., detecting the location of an analyte, e.g., abiological analyte) from a biological sample (e.g., present in abiological sample) that include: (a) optionally staining and/or imaginga biological sample on a substrate; (b) permeabilizing (e.g., providinga solution comprising a permeabilization reagent to) the biologicalsample on the substrate; (c) contacting the biological sample with anarray comprising a plurality of capture probes, wherein a capture probeof the plurality captures the biological analyte; and (d) analyzing thecaptured biological analyte, thereby spatially detecting the biologicalanalyte; where the biological sample is not removed from the substrate.

In some embodiments, provided herein are methods for spatially detectinga biological analyte of interest from a biological sample that include:(a) staining and imaging a biological sample on a substrate; (b)providing a solution comprising a permeabilization reagent to thebiological sample on the substrate; (c) contacting the biological samplewith an array on a substrate, wherein the array comprises one or morecapture probe pluralities thereby allowing the one or more pluralitiesof capture probes to capture the biological analyte of interest; and (d)analyzing the captured biological analyte, thereby spatially detectingthe biological analyte of interest; where the biological sample is notremoved from the substrate.

In some embodiments, the method further includes subjecting a region ofinterest in the biological sample to spatial transcriptomic analysis. Insome embodiments, one or more of the capture probes includes a capturedomain. In some embodiments, one or more of the capture probes comprisesa unique molecular identifier (UMI). In some embodiments, one or more ofthe capture probes comprises a cleavage domain. In some embodiments, thecleavage domain comprises a sequence recognized and cleaved by auracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease (APE1),U uracil-specific excision reagent (USER), and/or an endonuclease VIII.In some embodiments, one or more capture probes do not comprise acleavage domain and is not cleaved from the array.

In some embodiments, a capture probe can be extended (an “extendedcapture probe,” e.g., as described herein). For example, extending acapture probe can include generating cDNA from a captured (hybridized)RNA. This process involves synthesis of a complementary strand of thehybridized nucleic acid, e.g., generating cDNA based on the captured RNAtemplate (the RNA hybridized to the capture domain of the captureprobe). Thus, in an initial step of extending a capture probe, e.g., thecDNA generation, the captured (hybridized) nucleic acid, e.g., RNA, actsas a template for the extension, e.g., reverse transcription, step.

In some embodiments, the capture probe is extended using reversetranscription. For example, reverse transcription includes synthesizingcDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), usinga reverse transcriptase. In some embodiments, reverse transcription isperformed while the tissue is still in place, generating an analytelibrary, where the analyte library includes the spatial barcodes fromthe adjacent capture probes. In some embodiments, the capture probe isextended using one or more DNA polymerases.

In some embodiments, a capture domain of a capture probe includes aprimer for producing the complementary strand of a nucleic acidhybridized to the capture probe, e.g., a primer for DNA polymeraseand/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA,molecules generated by the extension reaction incorporate the sequenceof the capture probe. The extension of the capture probe, e.g., a DNApolymerase and/or reverse transcription reaction, can be performed usinga variety of suitable enzymes and protocols.

In some embodiments, a full-length DNA (e.g., cDNA) molecule isgenerated. In some embodiments, a “full-length” DNA molecule refers tothe whole of the captured nucleic acid molecule. However, if a nucleicacid (e.g., RNA) was partially degraded in the tissue sample, then thecaptured nucleic acid molecules will not be the same length as theinitial RNA in the tissue sample. In some embodiments, the 3′ end of theextended probes, e.g., first strand cDNA molecules, is modified. Forexample, a linker or adaptor can be ligated to the 3′ end of theextended probes. This can be achieved using single stranded ligationenzymes such as T4 RNA ligase or Circligase™ (available from Lucigen,Middleton, Wis.). In some embodiments, template switchingoligonucleotides are used to extend cDNA in order to generate afull-length cDNA (or as close to a full-length cDNA as possible). Insome embodiments, a second strand synthesis helper probe (a partiallydouble stranded DNA molecule capable of hybridizing to the 3′ end of theextended capture probe), can be ligated to the 3′ end of the extendedprobe, e.g., first strand cDNA, molecule using a double strandedligation enzyme such as T4 DNA ligase. Other enzymes appropriate for theligation step are known in the art and include, e.g., Tth DNA ligase,Taq DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNAligase, New England Biolabs), Ampligase™ (available from Lucigen,Middleton, Wis.), and SplintR (available from New England Biolabs,Ipswich, Mass.). In some embodiments, a polynucleotide tail, e.g., apoly(A) tail, is incorporated at the 3′ end of the extended probemolecules. In some embodiments, the polynucleotide tail is incorporatedusing a terminal transferase active enzyme.

In some embodiments, double-stranded extended capture probes are treatedto remove any unextended capture probes prior to amplification and/oranalysis, e.g., sequence analysis.

This can be achieved by a variety of methods, e.g., using an enzyme todegrade the unextended probes, such as an exonuclease enzyme, orpurification columns.

In some embodiments, extended capture probes are amplified to yieldquantities that are sufficient for analysis, e.g., via DNA sequencing.In some embodiments, the first strand of the extended capture probes(e.g., DNA and/or cDNA molecules) acts as a template for theamplification reaction (e.g., a polymerase chain reaction).

In some embodiments, the amplification reaction incorporates an affinitygroup onto the extended capture probe (e.g., RNA-cDNA hybrid) using aprimer including the affinity group. In some embodiments, the primerincludes an affinity group and the extended capture probes includes theaffinity group. The affinity group can correspond to any of the affinitygroups described previously.

In some embodiments, the extended capture probes including the affinitygroup can be coupled to a substrate specific for the affinity group. Insome embodiments, the substrate can include an antibody or antibodyfragment. In some embodiments, the substrate includes avidin orstreptavidin and the affinity group includes biotin. In someembodiments, the substrate includes maltose and the affinity groupincludes maltose-binding protein. In some embodiments, the substrateincludes maltose-binding protein and the affinity group includesmaltose. In some embodiments, amplifying the extended capture probes canfunction to release the extended probes from the surface of thesubstrate, insofar as copies of the extended probes are not immobilizedon the substrate.

In some embodiments, the extended capture probe or complement oramplicon thereof is released. The step of releasing the extended captureprobe or complement or amplicon thereof from the surface of thesubstrate can be achieved in a number of ways. In some embodiments, anextended capture probe or a complement thereof is released from thearray by nucleic acid cleavage and/or by denaturation (e.g., by heatingto denature a double-stranded molecule).

In some embodiments, the extended capture probe or complement oramplicon thereof is released from the surface of the substrate (e.g.,array) by physical means. For example, where the extended capture probeis indirectly immobilized on the array substrate, e.g., viahybridization to a surface probe, it can be sufficient to disrupt theinteraction between the extended capture probe and the surface probe.Methods for disrupting the interaction between nucleic acid moleculesinclude denaturing double stranded nucleic acid molecules are known inthe art. A straightforward method for releasing the DNA molecules (i.e.,of stripping the array of extended probes) is to use a solution thatinterferes with the hydrogen bonds of the double stranded molecules. Insome embodiments, the extended capture probe is released by an applyingheated solution, such as water or buffer, of at least 85° C., e.g., atleast 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C. In some embodiments,a solution including salts, surfactants, etc. that can furtherdestabilize the interaction between the nucleic acid molecules is addedto release the extended capture probe from the substrate.

In some embodiments, where the extended capture probe includes acleavage domain, the extended capture probe is released from the surfaceof the substrate by cleavage. For example, the cleavage domain of theextended capture probe can be cleaved by any of the methods describedherein. In some embodiments, the extended capture probe is released fromthe surface of the substrate, e.g., via cleavage of a cleavage domain inthe extended capture probe, prior to the step of amplifying the extendedcapture probe.

In some embodiments, probes complementary to the extended capture probecan be contacted with the substrate. In some embodiments, the biologicalsample can be in contact with the substrate when the probes arecontacted with the substrate. In some embodiments, the biological samplecan be removed from the substrate prior to contacting the substrate withprobes. In some embodiments, the probes can be labeled with a detectablelabel (e.g., any of the detectable labels described herein). In someembodiments, probes that do not specially bind (e.g., hybridize) to anextended capture probe can be washed away. In some embodiments, probescomplementary to the extended capture probe can be detected on thesubstrate (e.g., imaging, any of the detection methods describedherein).

In some embodiments, probes complementary to an extended capture probecan be about 4 nucleotides to about 100 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 10 nucleotides to about 90nucleotides long. In some embodiments, probes (e.g., detectable probes)complementary to an extended capture probe can be about 20 nucleotidesto about 80 nucleotides long. In some embodiments, probes (e.g.,detectable probes) complementary to an extended capture probe can beabout 30 nucleotides to about 60 nucleotides long. In some embodiments,probes (e.g., detectable probes) complementary to an extended captureprobe can be about 40 nucleotides to about 50 nucleotides long. In someembodiments, probes (e.g., detectable probes) complementary to anextended capture probe can be about 5, about 6, about 7, about 8, about9, about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, about 25, about 26, about 27, about 28, about 29, about 30,about 31, about 32, about 33, about 34, about 35, about 36, about 37,about 38, about 39, about 40, about 41, about 42, about 43, about 44,about 45, about 46, about 47, about 48, about 49, about 50, about 51,about 52, about 53, about 54, about 55, about 56, about 57, about 58,about 59, about 60, about 61, about 62, about 63, about 64, about 65,about 66, about 67, about 68, about 69, about 70, about 71, about 72,about 73, about 74, about 75, about 76, about 77, about 78, about 79,about 80, about 81, about 82, about 83, about 84, about 85, about 86,about 87, about 88, about 89, about 90, about 91, about 92, about 93,about 94, about 95, about 96, about 97, about 98, and about 99nucleotides long.

In some embodiments, about 1 to about 100 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 1 to about 10 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 10 to about 100 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 20 to about 90 probescan be contacted to the substrate and specifically bind (e.g.,hybridize) to an extended capture probe. In some embodiments, about 30to about 80 probes (e.g., detectable probes) can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe. In some embodiments, about 40 to about 70 probes can be contactedto the substrate and specifically bind (e.g., hybridize) to an extendedcapture probe. In some embodiments, about 50 to about 60 probes can becontacted to the substrate and specifically bind (e.g., hybridize) to anextended capture probe. In some embodiments, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50, about 51, about 52, about 53, about54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about68, about 69, about 70, about 71, about 72, about 73, about 74, about75, about 76, about 77, about 78, about 79, about 80, about 81, about82, about 83, about 84, about 85, about 86, about 87, about 88, about89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, and about 99 probes can be contacted to thesubstrate and specifically bind (e.g., hybridize) to an extended captureprobe.

In some embodiments, the probes can be complementary to a single analyte(e.g., a single gene). In some embodiments, the probes can becomplementary to one or more analytes (e.g., analytes in a family ofgenes). In some embodiments, the probes (e.g., detectable probes) can befor a panel of genes associated with a disease (e.g., cancer,Alzheimer's disease, Parkinson's disease).

In some instances, the ligated probe and capture probe can be amplifiedor copied, creating a plurality of cDNA molecules. In some embodiments,cDNA can be denatured from the capture probe template and transferred(e.g., to a clean tube) for amplification, and/or library construction.The spatially-barcoded cDNA can be amplified via PCR prior to libraryconstruction. The cDNA can then be enzymatically fragmented andsize-selected in order to optimize for cDNA amplicon size. P5 and P7sequences directed to capturing the amplicons on a sequencing flowcell(Illumina sequencing instruments) can be appended to the amplicons, i7,and i5 can be used as sample indexes, and TruSeq Read 2 can be added viaEnd Repair, A-tailing, Adaptor Ligation, and PCR. The cDNA fragments canthen be sequenced using paired-end sequencing using TruSeq Read 1 andTruSeq Read 2 as sequencing primer sites. The additional sequences aredirected toward Illumina sequencing instruments or sequencinginstruments that utilize those sequences; however a skilled artisan willunderstand that additional or alternative sequences used by othersequencing instruments or technologies are also equally applicable foruse in the aforementioned methods.

In some embodiments, where a sample is barcoded directly viahybridization with capture probes or analyte capture agents hybridized,bound, or associated with either the cell surface, or introduced intothe cell, as described above, sequencing can be performed on the intactsample.

A wide variety of different sequencing methods can be used to analyzebarcoded analyte (e.g., the ligation product). In general, sequencedpolynucleotides can be, for example, nucleic acid molecules such asdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), includingvariants or derivatives thereof (e.g., single stranded DNA or DNA/RNAhybrids, and nucleic acid molecules with a nucleotide analog).

Sequencing of polynucleotides can be performed by various systems. Moregenerally, sequencing can be performed using nucleic acid amplification,polymerase chain reaction (PCR) (e.g., digital PCR and droplet digitalPCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-basedsingle plex methods, emulsion PCR), and/or isothermal amplification.Non-limiting examples of methods for sequencing genetic materialinclude, but are not limited to, DNA hybridization methods (e.g.,Southern blotting), restriction enzyme digestion methods, Sangersequencing methods, next-generation sequencing methods (e.g.,single-molecule real-time sequencing, nanopore sequencing, and Polonysequencing), ligation methods, and microarray methods.

(1) Kits

In some embodiments, also provided herein are kits that include one ormore reagents to detect one or more analytes described herein. In someinstances, the kit includes a substrate comprising a plurality ofcapture probes comprising a spatial barcode and the capture domain. Insome instances, the kit includes a plurality of probes (e.g., a firstprobe, a second probe, one or more spanning probes, and/or a thirdoligonucleotide).

A non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) a substrate comprising a plurality ofcapture probes comprising a spatial barcode and a capture domain; (b) asystem comprising: a plurality of first probes and second probes,wherein a first probe and a second probe each comprises sequences thatare substantially complementary to an analyte, and wherein the secondprobe comprises a capture binding domain; and (c) instructions forperforming the method of any one of the preceding claims.

Another non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) an array comprising a plurality ofcapture probes; (b) a plurality of probes comprising a first probe and asecond, wherein the first probe and the second probe are substantiallycomplementary to adjacent sequences of an analyte, wherein the secondprobe comprises (i) a capture probe binding domain that is capable ofbinding to a capture domain of the capture probe and (ii) a linkersequence; (c) a plurality of enzymes comprising a ribonuclease and aligase; and (d) instructions for performing the method of any one of thepreceding claims.

Another non-limiting example of a kit used to perform any of the methodsdescribed herein includes: (a) an array comprising a plurality ofcapture probes; (b) a plurality of probes comprising a first probe and asecond probe, wherein the first probe and the second probe aresubstantially complementary to adjacent sequences of an analyte, whereinthe first probe includes a linker sequence, wherein the second probecomprises a capture probe binding domain that is capable of binding to acapture domain of the capture probe; (c) a plurality of enzymescomprising a ribonuclease and a ligase; and (d) instructions forperforming the method of any one of the preceding claims.

In some embodiments of any of the kits described herein, the kitincludes a second probe that includes a preadenylated phosphate group atits 5′ end and a first probe comprising at least two ribonucleic acidbases at the 3′ end.

EXAMPLES Example 1. Spatial Gene Expression Analysis of FFPE-FixedSamples Using RNA-Templated Ligation

Others have demonstrated in situ ligation. See Credle et al., NucleicAcids Research, Volume 45, Issue 14, 21 Aug. 2017, Page e128,https://doi.org/10.1093/nar/gkx471 (2017). However, the previousapproaches have utilized hybridization using poly(A) tails which led tooff-target binding. Here, the poly(A) tail on one probe oligonucleotidewas switched to another sequence.

As an overview, a non-limiting example of RNA-templated ligation on anFFPE-fixed sample was performed as described in FIG. 13. FFPE-fixedsamples were deparaffinized, stained (e.g., H&E stain), and imaged 1301.Samples were destained (e.g., using HCl) and decrosslinked 1302.Following decrosslinking, samples treated with pre-hybridization buffer(e.g., hybridization buffer without the first and second probes), probeswere added to the sample, probes hybridized, and samples were washed1303. Ligase was added to the samples to ligate hybridized probes togenerate a ligation product and samples were then washed 1304. Probeswere released from the analyte by contacting the biological sample withRNAse H 1305. Samples were then permeabilized to facilitate capture ofthe ligation product by the capture probes on the substrate 1306.Ligation products that hybridized to the capture probes were thenextended 1307. The extended capture probes were denatured 1308.Denatured, extended capture probes were indexed and the amplifiedlibraries were subjected to quality control 1309 before being sequenced.

FFPE sectioned mouse brain tissue slides were deparaffinized,permeabilized with PBST, and equilibrated with a pre-Hyb buffer twicefor 5 minutes each. Decrosslinking the sample was performed using eithera TE decrosslinking reagent or a PBS-tween decrosslinking reagent. ForTE decrosslinking, 100 μl of TE buffer (pH 9.0) (Genemed 10-0046) wasadded per sample. Samples treated with TE were subjected to a thermalcycler protocol according to Table 1.

TABLE 1 Decrosslinking Thermal Cycler Protocol Decrosslinking: Lid Temp:70 C., Volume: 100 uL Step Temp Time Pre-heat 70° C. ∞ Decrosslinking70° C. 60 minutes Hold 22° C. ∞

Following the thermal cycler protocol, slides were placed into a metalcassette and subject to the methods described in Table 2.

TABLE 2 Decrosslinking with HCl* Step Timing 1. Wash 1: Added 100 μl0.1N HCl 1 minute 2. Wash 2: Removed the HCl and add 100 μl HCl 1 minute3. Wash 3: Removed the HCl and add 100 μl HCl 1 minute 4. Bufferexchange: Removed HCl and add 100 μl TE buffer (pH 9.0) 5. Removed TEand add 100 μl TE (pH 9.0). 6. Incubated at 70° C. 1 hour 7. Removed TEand add 100 μl 1x PBS-Tween (0.05%) 8. Incubated at room temperature 15minutes *Note: All liquid was removed at each step.

RTL probes were designed to hybridize to adjacent sequences of eachanalyte (e.g., mRNA sequence) of interest in the genome, includingestrogen receptor, progesterone receptor, and ERBB2, also known as HER2.Here, 20,056 probe pairs (e.g., RHS and LHS probes) were added to eachtissue sample to capture 19,490 different genes. Two RTL probes (aleft-hand side (LHS) probe and a right-hand side (RHS) probe (see e.g.,FIG. 6)) for each analyte were added simultaneously and hybridized atadjacent sequences of the target mRNA, forming RNA:DNA duplexstructures. Following decrosslinking, the PBS-Tween was removed and theDNA probes (1 nM of each probe) were added to the tissue samples in ahybridization buffer for hybridizing the DNA probes to their respectivemRNA targets. The hybridization buffer included SSC, formamide or anequivalent, yeast tRNA as carrier and the RHS and LHS DNA probes.

One probe oligonucleotide (e.g., the RHS probe or the 3′ probe)comprises a non-target functional sequence at its 5′ end while the otherprobe oligonucleotide (e.g., the LHS probe or the 5′ probe) comprises anon-target polyA sequence at their 3′ ends. The DNA probes were added tothe tissue samples and incubated at 50° C. for 2 hours and 30 minutesaccording to thermal cycler protocol described in Table 3.

TABLE 3 Hybridization Protocol Hybridization Thermal Cycler ProtocolHyb: Lid Temp: 50 C., Volume: 100 uL Step Temp Time Pre-heat 50° C. ∞Hyb 50° C. 2 hours and 30 minutes Hold for post hyb washes 50° C. ∞ Hold22° C. ∞

Following incubation at 50° C. for 2½ hours, the hybridization bufferwas removed and the tissue was washed twice with each wash including 5minutes at 50° C. with post-hybridization wash buffer pre-heated to 50°C. Post-hybridization wash buffer (Post-hyb buffer) included: SSC, yeasttRNA and nuclease free water.

To ligate two diribo probe oligonucleotides that adjacently hybridizedon the target mRNA as described above, each sample was incubated withRNL2 T4 DNA ligase reaction mixture (ligation mix). The ligase reactionmixture included: T4 RNA Ligase Buffer (NEB B0293S), RNL2 ligase andnuclease free water.

To ligate two DNA probe oligonucleotides that adjacently hybridized onthe target mRNAs as described above, SplintR (NEB) in a ligation bufferwas added to each sample. The ligase reaction mixture included: Tris-HCl(pH 7.5) MnCl₂ ATP, DTT, surrogate fluid, SplintR ligase (NEB), andnuclease free water per sample. The ligation and wash steps wereperformed as described in Table 4.

Following DNA probe ligation the tissue samples were washed twice for 5minutes at 60° C. in a SSC/formamide post ligation wash buffer.

TABLE 4 Ligation Protocol Probe Lisation Step Time Removed ALL Post-HybBuffer before adding Ligase Mix 1 h  Added Ligase Mix and Incubated at37° C. Removed post-ligation wash buffer and added 2X SSC**. Repeated 2XSSC wash. The cassette was cooled down to room temperature before addingRNAse Wash 1: Removed Ligase Mix, added post-ligation wash buffer*, 5min and incubated at 60° C. for 5 minutes Remaining wash buffer atheated to 60° C. Wash 2: Removed post-ligation wash buffer, addedpre-heated 5 min post-ligation buffer and incubated at 60° C. for 5minutes. *Post ligation wash buffer included (per sample): SSC,formamide or an equivalent, and nuclease free water.

RNase H was added to digest the RNA strand of the hybridized RNA:DNAduplex. Briefly, the RNA of the DNA:RNA hybrids was digested byincubating the samples with RNase H mix for 30 minutes at 37° C., wherethe RNAse H mix included: RNAse H buffer and RNaseH. Following theincubation and while the same remained at 37° C., the biological samplewas permeabilized to release the ligated RTL probes using 1.25 mg/mLProteinase K. In particular, the Proteinase K solution included (persample): Tris (pH 7.5), MgCl2, Sarkosyl, or SDS, Proteinase K (Enzyme),and nuclease free water. The sample was incubated at 37° C. for at least5 minutes in the Proteinase K solution. The samples were then washedthree times with 2×SSC.

The released, ligated DNA probes that served as a proxy for the targetmRNA were allowed to hybridize to the capture domain on the captureprobe immobilized on the spatial array via the polyA tail on the 3′ endof the RHS probe. The captured ligated probes were copied, using thecapture probe as a template and the extension product was released fromthe spatial array. Briefly, the tissues were incubated with a secondstrand extension mix comprising Kapa Hifi DNA polymerase for 25 minutesat 53° C. Following incubation, the second strand extension mix wasremoved from the tissues and the tissues were washed with 2×SSC. Asolution of KOH was added to each of the tissue wells, the tissues wereincubated at room temperature for 10 minutes to release the extensionproduct from the spatial array and the supernatant from each tissue wellwas transferred for quantification, and library preparation. Samplequantification was performed using qPCR and KAPA SYBR FAST qPCR mastermix according to the manufacturer's instructions. Briefly, KAPA SYBRmaster mix was prepared by adding qPCR primer cDNA_F and qPCR primersRNA_R2. The thermal cycler protocol included: 3 minutes at 98° C.; 30cycles with 5 seconds at 98° C., and 30 seconds at 63° C. For librarypreparation, samples were indexed using an Amp Mix that included dualindexing primers and an Amp Mix. Nucleic acids were then sequenced andanalyzed.

As controls, concurrent experiments were run in which no ligase wasadded, pepsin was added before RNAse H treatment, or pepsin was addedafter RNAse H treatment.

As shown in FIGS. 14-19, spatial expression of ligated probe pairshighlights underlying mouse brain tissue physiology. FIG. 14 shows PCRresults demonstrating that a ligation product was captured andamplifiable whether the permeabilization occurred before or after RNaseH treatment (positive control=standard, negative control=minus ligase).Arrow 1 points to a PCR band that represents desired ligation products,whereas arrows 2, 3 and 4 represent non-ligation products. FIG. 15 showsnon-specific probe detected as a fraction of total reads for eachcondition. FIGS. 16A-B shows most probe combinations are specific. Thearrows indicate RHS probes that had increased background and thereforeinclude some non-specific hybridization. Looking at total counts in FIG.17, gene-specific LHS+RHS probes overlay with the tissue footprint (seeFIGS. 17A-C with black circle indicating tissue footprint).

Further, probes specific for various genes (e.g., Grp88, Penk, Plp1,Nptrx, and Mpb2) were added to the sample in Hybridization buffer firstfor 30 minutes at 60° C., and then for 2 hours at 45° C. (See, e.g.,FIG. 18 and FIG. 19). When target-specific UMIs were counted, a moredetailed expression pattern was observed. See, e.g., FIGS. 19B-19F. Thisanalysis revealed that longer hybridization led to higher sensitivity.Referring to FIGS. 18A-18E, white circles indicate spots reportingexpression for the indicated gene.

An overnight hybridization was performed comparative to a 2 hourhybridization as discussed. Analysis on mouse brain samples using 21,833probe pairs (e.g., RHS and LHS probes) added to each tissue sample tocapture 21,604 different genes revealed that overnight hybridization,thus longer hybridization, led to higher sensitivity compared to apositive control (see FIGS. 20A-20B). The mouse probes were designedfrom targets derived from Appris (see Rodriguez et al., Nucleic AcidsResearch, 46: D213-217, doi: 10.1093/nar/gkx997 (2018), which is hereinincorporated by reference in its entirety) and GENCODE. All probe pairswere non-overlapping and include generally about 1 probe pair per gene.

Example 2. Spatial Gene Expression Analysis of Triple Positive BreastCancer (TPBC) Using RNA Templated Ligation (RTL)

This example demonstrates that RTL can be performed on a sample in orderto identify analyte abundance and spatial location in an unbiasedmanner.

A two-year old triple positive (“TPBC,” HER2, estrogen receptor, andprogesterone receptor-positive) breast cancer sample preserved by FFPEprocessing was examined for analyte abundance and spatial location. TheTPBC samples were queried with DNA probes via RNA-templated ligationmethods. Before the ligation step, the TPBC tissue samples weredeparaffinized and stained per established protocols. For example, FFPETPBC tissue samples were prewarmed in a water bath (40° C.), sectioned(10 μm), dried at 42° C. for several hours and placed in a desiccator atroom temperature overnight. The dry, sectioned tissues weredeparaffinized by baking at 60° C., moved through a series of xylene andEtOH washes, rinsed in water several times. Following rinsing, thedeparaffinized tissues were stained with hematoxylin per establishedprotocols. The stained tissues were imaged, identifying regions of tumorand stroma. See FIG. 21A.

The tissues were decrosslinked to remove formaldehyde crosslinks withinthe sample thereby releasing the analytes for RNA templated ligation.Briefly, the tissue samples were incubated with an HCl solution for 1minute, repeated twice for a total of 3 minutes. Following HClincubations, the tissue sections were incubated at 70° C. for 1 hour inTE pH 9.0. TE was removed and the tissues were incubation in 1×PBS-Tweenfor 15 minutes.

RTL probes were designed to hybridize to adjacent sequences of eachanalyte (e.g., mRNA sequence) of interest in the genome, includingestrogen receptor, progesterone receptor, and ERBB2, also known as HER2.

Probes were designed from targets derived from Appris (see, Rodriguez etal., Nucleic Acids Research, 46: D213-217, doi: 10.1093/nar/gkx997(2018), which is herein incorporated by reference in its entirety) andGENCODE. All probe pairs were non-overlapping and include generallyabout 1 probe pair per gene.

Here, 20,056 probe pairs (e.g., RHS and LHS probes) were added to eachtissue sample to capture 19,490 different genes, including ESR1, PGR andHER2, in the human genome. Two RTL probes (a left-hand side (LHS) probeand a right-hand side (RHS) probe (see, e.g., FIG. 6)) for each analytewere added simultaneously and hybridized at adjacent sequences of thetarget mRNA, forming RNA:DNA duplex structures.

Following decrosslinking, the DNA probes (1 nm of each probe) were addedto the tissue samples in a hybridization buffer for hybridizing the DNAprobes to their respective mRNA targets. One probe oligonucleotide(e.g., the RHS probe or the 3′ probe) comprises a non-target functionalsequence at its 5′ end while the other probe oligonucleotide (e.g., theLHS probe or the 5′ probe) comprise a non-target polyA sequence at their3′ ends. Briefly, hybridization buffer with the DNA probes was added tothe tissue samples and the tissues were incubated at 50° C. ofapproximately 2½ hrs. The hybridization/DNA probe buffer was removed andthe tissues washed by addition of a post hybridization buffer withoutDNA probes and incubation at 50° C. for 5 minutes, for a total of 3 posthybridization washes.

To ligate the two DNA probe oligonucleotides that adjacently hybridizedon the target mRNAs as described above, SplintR (NEB) in a ligationbuffer was added to each tissue sample and the tissues were incubated at37° C. for 60 minutes. Following DNA probe ligation the tissue sampleswere washed twice for 5 minutes at 60° C. in a SSC/formamide postligation wash buffer.

Next, RNase H was added to digest the RNA strand of the hybridizedRNA:DNA duplex. Briefly, the RNA of the DNA:RNA hybrids was digested byincubating the tissues with RNase H for 30 minutes at 37° C. Thebiological sample then was permeabilized to release the ligated RTLprobes and contacted with a plurality of capture probes attached to aslide. In particular, after 30 minutes, the tissues were washed andpermeabilized by adding 1.25 mg/ml Proteinase K, incubated at 37° C. forat least 5 minutes and then washed to remove the protease.

The released, ligated DNA probes that served as a proxy for the targetmRNA were allowed to hybridize to the capture domain on the captureprobe (capture probes immobilized on the spatial array) via the polyAtail on the 3′ end of the RHS probe. The captured ligated probes werecopied, using the capture probe as a template and the extension productwas released from the spatial array. Briefly, the tissues were incubatedwith a second strand extension mix comprising Kapa Hifi DNA polymerase(Roche) for 25 minutes at 53° C. Following incubation, the extension mixwas removed from the tissues and the tissues were washed with SSC. Asolution of KOH was added to each of the tissue wells, the tissues wereincubated at room temperature for 10 minutes to release the extensionproduct from the spatial array and the supernatant from each tissue wellwas transferred for quantitation, library preparation and sequencing onthe Illumina NextSeq sequencing instrument.

Assessing the whole transcriptome helped to better understand TPBCheterogeneity. For example, it was observed that using 20,056 probepairs (e.g., RHS and LHS probes) to capture 19,490 different genesrevealed comparable results to a positive control in terms of number ofUMIs per cell versus number of genes per cell (FIG. 21B). Analysis ofspatial location and abundance of each RTL ligated probe that hybridizedto the array revealed eight (8) different clusters of expression,demonstrating that differential gene expression and location of theexpressed genes can be determined using RTL probes. See FIGS. 21C-21D.Referring to FIG. 21C, each point on the plot is a spot on the array.Each spot is assigned to a cluster, which are indicated by blackcircles. In some cases, spots assigned to a cluster are outside theindicated circle on the plot. Referring to FIG. 21D, each spot on thearray is assigned to a cluster and each cluster is indicated, in part,by circles. In some cases, spots are assigned to a cluster but are notwithin the indicated circles on the array. Finally, individual analyteexpression was determined. A TPBC sample routinely exhibited elevatedlevels of estrogen receptor, progesterone receptor, and ERRB2 (HER2). Asshown in FIGS. 21E-21GA.

Embodiment A1. A method for determining a location of an analyte in abiological sample comprising:

(a) contacting the biological sample with an array comprising aplurality of capture probes, wherein a capture probe of the pluralitycomprises: (i) a spatial barcode and (ii) a capture domain;

(b) contacting the biological sample with a first probe oligonucleotideand a second probe oligonucleotide, wherein the first probeoligonucleotide and the second probe oligonucleotide each comprise asequence that is substantially complementary to adjacent sequences ofthe analyte, and wherein the second probe oligonucleotide comprises acapture probe capture domain;

(c) hybridizing the first probe oligonucleotide and the second probeoligonucleotide to the analyte in a formamide-free hybridization buffer;

(d) ligating the first probe oligonucleotide and the second probeoligonucleotide, thereby generating a ligation product;

(e) releasing the ligated product from the analyte and hybridizing theligation product to the capture domain; and

(f) determining (i) all or a part of the sequence of the ligationproduct specifically bound to the capture domain, or a complementthereof, and (ii) all or a part of the sequence of the spatial barcode,or a complement thereof, and using the determined sequence of (i) and(ii) to identify the location of the analyte in the biological sample.

Embodiment A2. The method of Embodiment A1, wherein the capture probefurther comprises one or more functional domains, a unique molecularidentifier, a cleavage domain, and combinations thereof.

Embodiment A3. The method of Embodiment A1 or A2, wherein the arraycomprises one or more features on a substrate.

Embodiment A4. The method of Embodiment A3, wherein the one or morefeatures comprises a bead.

Embodiment A5. The method of Embodiment A3, wherein the substratecomprises a slide.

Embodiment A6. The method of any one of Embodiments A1-A5, wherein theformamide-free hybridization buffer is a saline-sodium citrate (SSC)hybridization buffer.

Embodiment A7. The method of Embodiment A6, wherein SSC is present inthe SSC hybridization buffer from about 1×SSC to about 6×SSC.

Embodiment A8. The method of Embodiments A6 or A7, wherein SSC ispresent in the SSC hybridization buffer from about 2×SSC to about 4×SSC.

Embodiment A9. The method of any one of Embodiments A6-A8, wherein theSSC hybridization buffer comprises a solvent.

Embodiment A10. The method of Embodiment A9, wherein the solventcomprises ethylene carbonate.

Embodiment A11. The method of any one of Embodiments A6-A10, whereinethylene carbonate is present in the SSC hybridization buffer from about10% (w/v) to about 25% (w/v).

Embodiment A12. The method of any one of Embodiments A6-A11, whereinethylene carbonate is present in the SSC hybridization buffer from about15% (w/v) to about 20% (w/v).

Embodiment A13. The method of any one of Embodiments A6-A12, whereinethylene carbonate is present in the SSC hybridization buffer at about13% (w/v).

Embodiment A14. The method of any one of Embodiments A6-A13, wherein theSSC hybridization buffer is at a temperature from about 40° C. to about60° C.

Embodiment A15. The method of any one of Embodiments A6-A14, wherein theSSC hybridization buffer is at temperature from about 45° C. to about55° C. Embodiment A16. The method of any one Embodiments A6-A15, wherein the SSC hybridization buffer is at a temperature of about 50° C.

Embodiment A17. The method of any one of Embodiments A6-A16, wherein theSSC hybridization buffer further comprises one or more of a yeast tRNA,a crowder, or an additive.

Embodiment A18. The method of any one of Embodiments A1-A17, whereinligating in step (d) comprises a ligase.

Embodiment A19. The method of Embodiment A18, wherein the ligase is oneor more of a T4 RNA ligase (Rnl2), a SplintR ligase, a single strandedDNA ligase, or a T4 DNA ligase.

Embodiment A20. The method of Embodiment A19, wherein the ligase is a T4DNA ligase.

Embodiment A21. The method of any one of Embodiments A1-A20, furthercomprising removing one or more unhybridized first probeoligonucleotides, one or more unhybridized second probeoligonucleotides, or both, from the array.

Embodiment A22. The method of Embodiment A21, wherein the removingcomprises washing the one or more unhybridized first probeoligonucleotides, the one or more unhybridized second probeoligonucleotides, or both, from the array in a formamide-free washbuffer.

Embodiment A23. The method of Embodiment A21 or A22, wherein theformamide-free wash buffer is an SSC wash buffer.

Embodiment A24. The method of any one of Embodiments A21-A23, whereinSSC is present in the SSC wash buffer from about 0.01×SSC to about1×SSC.

Embodiment A25. The method of any one of Embodiments A21-A24, whereinSSC is present in the SSC wash buffer at about 0.1×SSC.

Embodiment A26. The method of any one of Embodiments A21-A25, whereinthe SSC wash buffer comprises a detergent.

Embodiment A27. The method of Embodiment A26, wherein the detergentcomprises sodium dodecyl sulfate (SDS).

Embodiment A28. The method of any one of Embodiments A21-A26, wherein

SDS is present in the SSC wash buffer from about 0.01% (v/v) to about0.5% (v/v).

Embodiment A29. The method of any one of Embodiments A21-A28, whereinthe SDS is present in the SSC wash buffer at about 0.1% (v/v).

Embodiment A30. The method of any one of Embodiments A21-A29, whereinthe SSC wash buffer comprises a solvent.

Embodiment A31. The method of any one of Embodiments A21-A30, whereinthe solvent comprises ethylene carbonate.

Embodiment A32. The method of any one of Embodiments A21-A31, whereinethylene carbonate is present in the SSC wash buffer from about 10%(w/v) to about 25% (w/v).

Embodiment A33. The method of any one of Embodiments A21-A32, whereinethylene carbonate is present in the SSC wash buffer from about 15%(w/v) to about 20% (w/v).

Embodiment A34. The method of any one of Embodiments A21-A33, whereinethylene carbonate is present in the SSC wash buffer at about 16% (w/v).

Embodiment A35. The method of any one of Embodiments A21-A34, whereinthe SSC wash buffer is at a temperature from about 50° C. to about 70°C.

Embodiment A36. The method of any one of Embodiments A21-A35, whereinthe SSC wash buffer is at temperature from about 55° C. to about 65° C.

Embodiment A37. The method of any one Embodiments A21-A36, where in theSSC wash buffer is at a temperature of about 60° C.

Embodiment A38. The method of any one of Embodiments A1-A37, whereinreleasing in step (e) comprises contacting the ligation product with anendoribonuclease.

Embodiment A39 The method of Embodiment A38, wherein theendoribonuclease is one or more of RNase H, RNase A, RNase C, or RNaseI.

Embodiment A40. The method of Embodiment A38 or A39, wherein theendoribonuclease is RNAse H.

Embodiment A41. The method of Embodiment A40, wherein the RNase Hcomprises RNase H1, RNase H2, or both.

Embodiment A42. The method of any one Embodiments A1-A41, furthercomprising extending a 3′ end of the capture probe using the ligationproduct as a template for an extension reaction.

Embodiment A43. The method of Embodiment A42, wherein extending the 3′end of the capture probe comprises reverse transcribing the analyte,thereby generating a sequence complementary to the analyte.

Embodiment A44. The method of Embodiment A43, wherein reversetranscribing the analyte comprises a reverse transcriptase.

Embodiment A45. The method of any of one of Embodiments A1-A44, whereinthe analyte is RNA.

Embodiment A46. The method of any of one of Embodiments A1-A45, whereinthe RNA is an mRNA.

Embodiment A47. The method of any one of Embodiments A1-A46, wherein thebiological sample is a tissue sample.

Embodiment A48. The method of Embodiment A47, wherein the tissue sampleis a tissue section.

Embodiment A49. The method of any one of Embodiments A1-A46, wherein thebiological sample a fresh frozen biological sample.

Embodiment A50. The method of any one of Embodiments A1-A46, wherein thebiological sample is a fixed biological sample.

Embodiment A51. The method of Embodiment A41, wherein the fixedbiological sample is a formalin-fixed paraffin-embedded sample.

Embodiment A52. The method of any one of Embodiments A1-A51, wherein themethod further comprises permeabilizing the biological sample.

Embodiment A53. The method of Embodiment A52, wherein permeabilizing thebiological sample occurs before releasing the ligation product from theanalyte.

Embodiment A54. The method of Embodiment A52 or A53, whereinpermeabilizing the biological sample comprises an endopeptidase.

Embodiment A55. The method of any one of Embodiments A1-A54, wherein themethod further comprises amplifying the ligation product prior tocontacting the biological sample with the array.

Embodiment A56. The method of any one of Embodiments A1-A55, whereindetermining in step (f) comprises sequencing.

Embodiment A57. The method of any one of Embodiments A1-A56, wherein themethod further comprises a capture probe capture domain blocking moietythat specifically binds the capture probe capture domain.

Embodiment A58. The method of any one of Embodiments A1-A57, furthercomprising releasing the capture probe capture domain blocking moietyfrom the capture probe capture domain prior to contacting the biologicalsample with the array.

Embodiment A59. The method of any one of Embodiments A1-A58, wherein thecapture probe capture domain comprises a homopolymeric sequence.

Embodiment A60. The method of Embodiment A59, wherein the capture probecapture domain comprises a poly(A) sequence.

Embodiment B

Embodiment B1. A method for identifying a location of an analyte in abiological sample, the method comprising:

(a) contacting the biological sample with a substrate comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a capture domain and a spatial barcode;

(b) contacting the biological sample with a first probe and a secondprobe, wherein a portion of the first probe and a portion of the secondprobe are substantially complementary to adjacent sequences of theanalyte,

wherein the first probe comprises a sequence that is substantiallycomplementary to a first target sequence of the analyte,

wherein the second probe comprises:

-   -   (i) a first sequence that is substantially complementary to a        second target sequence of the analyte;    -   (ii) a linker sequence;    -   (iii) a second sequence that is substantially complementary to a        third target sequence of the analyte; and    -   (iv) a capture probe capture domain that is capable of binding        to a capture domain of a capture probe;

(c) hybridizing the first probe and the second probe to the analyte;

(d) ligating the first probe and the second probe, thereby creating aligation product;

(e) releasing the ligation product from the analyte;

(f) hybridizing the capture probe capture domain to a capture domain;and

(g) determining (i) all or a part of the sequence of the ligationproduct specifically bound to the capture domain, or a complementthereof, and (ii) all or a part of the sequence of the spatial barcode,or a complement thereof, and using the determined sequence of (i) and(ii) to identify the location of the analyte in the biological sample.

Embodiment B2. The method of Embodiment B1, wherein the second probecomprises from 5′ to 3′: a first sequence, a linker sequence, a secondsequence, and a capture probe capture domain.

Embodiment B3. The method of any one of Embodiments B 1-B2, wherein thefirst target sequence of the analyte is directly adjacent to the secondtarget sequence of the analyte.

Embodiment B4. The method of any one of Embodiments B 1-B3, wherein thesecond target sequence is not directly adjacent to the third targetsequence on the analyte.

Embodiment B5. The method of any one of Embodiments B 1-B4, wherein thesecond target sequence and the third target sequence are on differentexons of the analyte.

Embodiment B6. The method of any one of Embodiments B 1-B4, wherein thesecond target sequence and the third target sequence are within the sameexon of the analyte but are not directly adjacent.

Embodiment B7. The method of any one of Embodiments B 1-B6, wherein thelinker sequence comprises a total of about 1 nucleotide to about 100nucleotides.

Embodiment B8. The method of Embodiment B7, wherein the linker furthercomprises a barcode sequence that serves as a proxy for identifying theanalyte.

Embodiment B9. A method for identifying a location of an analyte in abiological sample, the method comprising:

(a) contacting the biological sample with a substrate comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a capture domain and a spatial barcode;

(b) contacting the biological sample with a first probe and a secondprobe, wherein a portion of the first probe and a portion of the secondprobe are substantially complementary to adjacent sequences of theanalyte,

wherein the first probe comprises:

-   -   (i) a first sequence that is substantially complementary to a        first target sequence of the analyte;    -   (ii) a linker sequence;    -   (iii) a second sequence that is substantially complementary to a        second target sequence of the analyte; and

wherein the second probe comprises a sequence that is substantiallycomplementary to a third target sequence of the analyte and a captureprobe capture domain that is capable of binding to a capture domain of acapture probe;

(c) hybridizing the first probe and the second probe to the analyte;

(d) ligating the first probe and the second probe, thereby creating aligation product;

(e) releasing the ligation product from the analyte;

(f) hybridizing the capture probe capture domain to a capture domain;and

(g) determining (i) all or a part of the sequence of the ligationproduct specifically bound to the capture domain, or a complementthereof, and (ii) all or a part of the sequence of the spatial barcode,or a complement thereof, and using the determined sequence of (i) and(ii) to identify the location of the analyte in the biological sample.

Embodiment B10. The method of Embodiment F9, wherein the second targetsequence is directly adjacent to the third target sequence.

Embodiment B11. The method of any one of Embodiments B9-B10, wherein thefirst probe comprises from 5′ to 3′: a first sequence, a linkersequence, and a second sequence.

Embodiment B12. The method of any one of Embodiments B9-B11, wherein thefirst probe further comprises a functional sequence.

Embodiment B13. The method of Embodiment B13, wherein the functionalsequence is a primer sequence.

Embodiment B14. The method of Embodiment B12 or B13, wherein the firstprobe comprises from 5′ to 3′: a functional sequence, a first sequence,a linker sequence, and a second sequence.

Embodiment B15. The method of any one of Embodiments B9-B14, wherein thefirst target sequence is not directly adjacent to the second targetsequence on the analyte.

Embodiment B16. The method of Embodiment B15, wherein the first targetsequence and second target sequence of are on different exons.

Embodiment B17. The method of Embodiment B15, wherein the first targetsequence and the second target sequence are within the same exon but arenot directly adjacent.

Embodiment B18. The method of any one of Embodiments B9-B17, wherein thelinker sequence comprises a total of about 1 nucleotide to about 100nucleotides.

Embodiment B19. The method of Embodiment B18, wherein the linker furthercomprises a barcode sequence that serves as a proxy for identifying theanalyte.

Embodiment B20. A method for identifying a location of an analyte in abiological sample, the method comprising:

(a) contacting the biological sample with a substrate comprising aplurality of capture probes, wherein a capture probe of the plurality ofcapture probes comprises a capture domain and a spatial barcode;

(b) contacting the biological sample with a first probe, a second probe,and one or more spanning probes,

wherein the first probe is substantially complementary to a firstportion of the analyte,

wherein the second probe is substantially complementary to a secondportion of the analyte and further comprises a capture probe capturedomain, and

wherein the spanning probe comprises:

-   -   (i) a first sequence that is substantially complementary to a        first target sequence of the analyte, and    -   (ii) a second sequence that is substantially complementary to a        second target sequence of the analyte;

(c) hybridizing the first probe, the second probe, and the spanningprobe to the analyte;

(d) ligating the first probe, the one or more spanning probes, and thesecond probe, thereby creating a ligation product that is substantiallycomplementary to the analyte;

(e) releasing the ligation product from the analyte;

(f) hybridizing the capture probe capture domain to a capture domain;and

(g) determining (i) all or a part of the sequence of the ligationproduct specifically bound to the capture domain, or a complementthereof, and (ii) all or a part of the sequence of the spatial barcode,or a complement thereof, and using the determined sequence of (i) and(ii) to identify the location of the analyte in the biological sample.

Embodiment B21. The method of Embodiment B20, wherein the spanningoligonucleotide further comprises a functional sequence.

Embodiment B22. The method of Embodiment B20 or B, wherein the spanningoligonucleotides comprises from 5′ to 3′: a first sequence, a functionalsequence, and a second sequence.

Embodiment B23. The method of any one of Embodiments B20-B22, whereinthe functional sequences comprises a linker sequence.

Embodiment B24. The method of Embodiment B23, wherein the linkersequence comprises a total of about 1 nucleotides to about 100nucleotides.

Embodiment B25. The method of any one of Embodiments B20-B24, whereinthe functional sequences comprises a barcode sequence.

Embodiment B26. The method of Embodiment B25, wherein the barcodesequence comprises a sequence that serves as a proxy for identifying theanalyte.

Embodiment B27. The method of any one of Embodiments B20-B26, whereinthe functional sequence comprises one or more linker sequences and abarcode sequence.

Embodiment B28. The method of Embodiment B27, wherein the functionalsequence comprises a barcode sequence flanked by linker sequences.

Embodiment B29. The method of any one of Embodiments B20-B28, whereinthe linker sequence comprises a total of about 1 nucleotides to about100 nucleotides.

Embodiment B30. The method of any one of Embodiments B20-B29, whereinthe first sequence of the spanning probe and the second sequence of thespanning probe are substantially complementary to sequences within thesame exon.

Embodiment B31. The method of Embodiment B30, wherein the first targetsequence of the analyte and the second target of the analyte are locatedwithin the same exon.

Embodiment B32. The method of any one of Embodiments B20-B29, whereinthe first sequence of the spanning probe and the second sequence of thespanning probe are substantially complementary to sequences within thedifferent exons of the same gene.

Embodiment B33. The method of Embodiment B32, wherein the first targetsequence of the analyte and the second target sequence of the analyteare located on different exons of the same gene.

Embodiment B34. The method of any one of Embodiments B20-33, wherein thefirst portion of the analyte is directly adjacent to the first targetsequence, and/or wherein the second portion of the analyte is directlyadjacent to the second target sequence.

Embodiment B35. The method of any one of Embodiments B20-B34, whereinthe spanning probe comprises at least two ribonucleic acid based at the3′ end.

Embodiment B36. The method of any one of Embodiments B20-B35, whereinthe spanning probe comprises a phosphorylated nucleotide at the 5′ end.

Embodiment B37. The method of any one of Embodiments B20-B36, whereinthe one or more spanning probes comprises one spanning probe.

Embodiment B38. The method of any one of Embodiments B20-B36, whereinthe one or more spanning probes comprises at least two, at least three,at least four, at least five, or more spanning probes.

Embodiment B39. The method of Embodiment B38, wherein the one or morespanning probes comprise:

-   -   (i) a third sequence that is substantially complementary to a        third target sequence of the analyte, and    -   (ii) a fourth sequence that is substantially complementary to a        fourth target sequence of the analyte.

Embodiment B40. The method of Embodiment B39, wherein the first targetsequence is located in a first exon, the second target sequence islocated in a second exon, and the third target sequence and the fourthtarget sequence are located in a third exon.

Embodiment B41. The method of Embodiments B39, wherein the first targetsequence is located in a first exon, the second target sequence islocated in a second exon, and the third target sequence is located in athird exon, and the fourth target sequence is located in a fourth exon.

Embodiment B42. The method of any one of Embodiments B38-B41, whereinthe method comprises ligating:

-   -   the first probe to the spanning probe,    -   the spanning probe to one or more additional spanning probes,        and    -   the one or more additional spanning probes spanning        oligonucleotide to the second probe, thereby creating a ligation        product that is substantially complementary to the analyte.

Embodiment B43. The method of any one of Embodiments B38-B42, whereinthe one or more additional spanning probes oligonucleotide furthercomprises a functional sequence.

Embodiment B44. The method of Embodiment B43, wherein the functionalsequences comprises (i) a linker sequence, (ii) a barcode sequence, or(ii) one or more linkers and a barcode sequence.

Embodiment B45. The method of any one of Embodiments B38-B44, whereinthe one or more additional spanning probes comprises at least tworibonucleic acid based at the 3′ end.

Embodiment B46. The method of any one of Embodiments B38-B45, whereinthe one or more additional spanning probes comprises a phosphorylatednucleotide at the 5′ end.

Embodiment B47. The method of any one of Embodiments B20-B46, whereinthe first probe further comprises a functional sequence.

Embodiment B48. The method of Embodiment B47, wherein the functionalsequence is a primer sequence.

Embodiment B49. The method of any one of the preceding Embodiments,wherein the first probe comprises at least two ribonucleic acid bases atthe 3′ end.

Embodiment B50. The method of any one of the preceding Embodiments,wherein the second probe comprises a phosphorylated nucleotide at the 5′end.

Embodiment B51. The method of any one of the preceding Embodiments,wherein the method further comprises providing a capture probe capturedomain blocking moiety that interacts with the capture probe capturedomain.

Embodiment B52. The method of Embodiment B51, wherein the method furthercomprises releasing the capture probe capture domain blocking moietyfrom the capture probe capture domain prior to step (f).

Embodiment B53. The method of any one of the preceding Embodiments,wherein the capture probe capture domain comprises a poly-adenylated(poly(A)) sequence or a complement thereof.

Embodiment B54. The method of Embodiment B53, wherein the capture probecapture domain blocking moiety comprises a poly-uridine sequence, apoly-thymidine sequence, or both.

Embodiment B55. The method of Embodiment B52, wherein releasing thepoly-uridine sequence from the poly(A) sequence comprises denaturing theligation product or contacting the ligation product with an endonucleaseor exonuclease.

Embodiment B56. The method of any one of the preceding Embodiments,wherein the capture probe capture domain comprises a sequence that iscomplementary to all or a portion of the capture domain of the captureprobe.

Embodiment B57. The method of any one of the preceding Embodiments,wherein the capture probe capture domain comprises a degeneratesequence.

Embodiment B58. The method of any one of the preceding Embodiments,wherein the ligation step comprises using enzymatic ligation or chemicalligation.

Embodiment B59. The method of Embodiment B58, wherein the enzymaticligation utilizes a ligase.

Embodiment B60. The method of Embodiment B59, wherein the ligase is oneor more of a T4 RNA ligase (Rnl2), a splintR ligase, a single strandedDNA ligase, or a T4 DNA ligase.

Embodiment B61. The method of Embodiment B60, wherein the ligase is a T4RNA ligase 2 (Rnl2) ligase.

Embodiment B62. The method of any one of the preceding Embodiments,wherein the first probe, the second probe, and the one or more spanningprobes are DNA probes.

Embodiment B63. The method of Embodiment B62, wherein the steps (b) and(c) each creates a RNA: DNA hybrid.

Embodiment B64. The method of any one of the preceding Embodiments,wherein steps (b) and (c) are performed at substantially the same time.

Embodiment B65. The method of any one of the preceding Embodiments,wherein the biological sample comprises a FFPE sample.

Embodiment B66. The method of Embodiment B65, wherein the tissue sampleis the FFPE tissue sample, and the tissue sample is decrosslinked.

Embodiment B67. The method of any one of the preceding Embodiments,wherein the biological sample comprises a tissue section.

Embodiment B68. The method of any one of the preceding Embodiments,wherein the biological sample comprises a fresh frozen sample.

Embodiment B69. The method of any one of the preceding Embodiments,wherein the biological sample comprises live cells.

Embodiment B70. The method of any one of the preceding Embodiments,wherein the analyte comprises RNA and/or DNA.

Embodiment B71. The method of any one of the preceding Embodiments,wherein the analyte is RNA.

Embodiment B72. The method of Embodiment B71, wherein the RNA is anmRNA.

Embodiment B73. The method of any one of the preceding Embodiments,wherein the biological sample was previously stained.

Embodiment B74. The method of Embodiment B73, wherein the biologicalsample was previously stained using hematoxylin and eosin (H&E).

Embodiment B75. The method of Embodiment B73 or B74, wherein thebiological sample was previously stained using immunofluorescence orimmunohistochemistry.

Embodiment B76. The method of any one of the preceding Embodiments,wherein the method further comprises contacting the biological samplewith a permeabilization agent.

Embodiment B77. The method of any one of the preceding Embodiments,wherein the releasing step comprises removing the ligated probe from theanalyte.

Embodiment B78. The method of Embodiment B77, wherein the releasing stepcomprises contacting the ligated probe with an endoribonuclease.

Embodiment B79. The method of Embodiment B78, wherein theendoribonuclease is one or more of RNase H, RNase A, RNase C, or RNaseI.

Embodiment B80. The method of Embodiment B79, wherein the RNase Hcomprises RNase H1, RNase H2, or

RNase H1 and RNase H2.

Embodiment B81. The method of any one of the preceding Embodiments,wherein the determining step comprises amplifying all or part of theligation product specifically bound to the capture domain.

Embodiment B82. The method of Embodiment B81, wherein the amplifying isisothermal.

Embodiment B83. The method of Embodiment B81, wherein the amplifying isnot isothermal.

Embodiment B84. The method of any one of Embodiments B81-B83, wherein anamplifying product comprises (i) all or part of sequence of the ligationproduct specifically bound to the capture domain, or a complementthereof, and (ii) all or a part of the sequence of the spatial barcode,or a complement thereof.

Embodiment B85. The method of any one of the preceding Embodiments,wherein the determining step comprises sequencing.

Embodiment B86. The method of Embodiment B85, wherein the sequencingstep comprises in situ sequencing.

Embodiment B87. A kit comprising

(a) an array comprising a plurality of capture probes;

(b) a plurality of probes comprising a first probe and a secondoligonucleotide, wherein the first probe and the second probe aresubstantially complementary to adjacent sequences of an analyte, whereinthe second probe comprises (i) a capture probe capture domain that iscapable of binding to a capture domain of the capture probe and (ii) alinker sequence;

(c) a plurality of enzymes comprising a ribonuclease and a ligase; and

(d) an instruction for using the kit.

Embodiment B88. A kit comprising

(a) an array comprising a plurality of capture probes;

(b) a plurality of probes comprising a first probe and a secondoligonucleotide, wherein the first probe and the second probe aresubstantially complementary to adjacent sequences of an analyte, whereinthe first probe includes a linker sequence, wherein the second probecomprises a capture probe capture domain that is capable of binding to acapture domain of the capture probe;

(c) a plurality of enzymes comprising a ribonuclease and a ligase; and

(d) an instruction for using the kit.

Embodiment B89. A kit comprising:

(a) an array comprising a plurality of capture probes;

(b) a plurality of probes comprising a first probe and a secondoligonucleotide, wherein the second probe comprises a capture probecapture domain that is capable of binding to a capture domain of thecapture probe;

(c) a plurality of spanning probes, wherein a spanning probe of theplurality of spanning probes comprises a first sequence, a linkersequence, and a second sequence, wherein the first sequence of thespanning probe and the first probe are substantially complementary toadjacent sequences of an analyte, wherein the second sequence of thespanning probe and the second probe are substantially complementary toadjacent sequences of the analyte;

(d) a plurality of enzymes comprising a ribonuclease and a ligase; and

(e) an instruction for using the kit.

Embodiment B90. The kit of any one of Embodiments B87-B89, wherein theribonuclease is RNase H.

Embodiment B91. The kit of any one of Embodiments B87-B90, wherein theligase is one or more of a T4 RNA ligase (Rnl2), a splintR ligase, asingle stranded DNA ligase, or a T4 DNA ligase.

Embodiment B92. The kit of Embodiment B91, wherein the ligase is a T4RNA ligase 2 (Rnl2) ligase.

1. A composition comprising: (a) a spatial array comprising a pluralityof capture probes, wherein a capture probe of the plurality of captureprobes comprises a spatial barcode and a capture domain; (b) abiological sample on the spatial array wherein the biological samplecomprises a plurality of analytes; (c) a first probe and a second probethat are ligated together, wherein the first probe and the second probeeach comprise one or more sequences that are substantially complementaryto sequences of the analyte and wherein one of the first probe or thesecond probe comprises a capture probe capture domain that is hybridizedto the capture domain of the capture probe on the spatial array.
 2. Thecomposition of claim 1, wherein the first probe or the second probe thatdoes not comprise the capture probe capture domain comprises afunctional domain.
 3. The composition of claim 1, wherein the secondprobe comprises the capture probe capture domain.
 4. The composition ofclaim 1, wherein the first probe further comprises a primer sequence. 5.The composition of claim 1, wherein the first probe and the second probeare substantially complementary to adjacent sequences of the analyte. 6.The composition of claim 1, wherein the first probe and the second probeare substantially complementary to sequences that are not adjacent toeach other on the analyte, wherein one of the first probe or the secondprobe is extended with a DNA polymerase.
 7. The composition of claim 1,wherein the first probe comprises at least two ribonucleic acid bases atthe 3′ end, and wherein the second probe comprises a phosphorylatednucleotide at the 5′ end.
 8. The composition of claim 1, wherein thefirst probe and/or the second probe is a DNA probe.
 9. The compositionof claim 1, further comprising 5000 or more ligated probe pairs, whereina ligated probe pair of the 5000 or more ligated probe pairs comprisesthe first probe and the second probe that are ligated together, andwherein at least one of the ligated probe pairs is hybridized to thecapture domain of a capture probe on the spatial array.
 10. Thecomposition of claim 1, wherein prior to ligation to the second probethe first probe comprised a pre-adenylated phosphate group at its 5′end.
 11. The composition of claim 1, wherein the capture probe capturedomain comprises a poly-adenylated sequence or a complement thereof. 12.The composition of claim 1, wherein the capture probe capture domaincomprises a degenerate sequence.
 13. The composition of claim 1, whereinthe capture domain comprises a poly-uridine sequence or a poly-thymidinesequence.
 14. The composition of claim 1, wherein the analyte comprisesRNA.
 15. The composition of claim 14, wherein the RNA is mRNA.
 16. Thecomposition of claim 1, further comprising a DNA polymerase.
 17. Thecomposition of claim 1, further comprising a RNase H enzyme, wherein theRNase H enzyme comprises RNase H1, RNase H2, or both.
 18. Thecomposition of claim 1, further comprising a permeabilization agent. 19.The composition of claim 18, wherein the permeabilization agent isproteinase K or pepsin.
 20. The composition of claim 1, furthercomprising a ligase.
 21. The composition of claim 20, wherein the ligaseis one or more of a T4 RNA ligase (Rnl2), a splintR ligase, a singlestranded DNA ligase, or a T4 DNA ligase.
 22. The composition of claim 1,wherein the biological sample is a tissue sample.
 23. The composition ofclaim 22, wherein the tissue sample is a formalin-fixed,paraffin-embedded (FFPE) tissue sample, a fresh tissue sample, or afrozen tissue sample.
 24. The composition of claim 23, wherein thetissue sample is the FFPE tissue sample.
 25. The composition of claim 1,wherein the biological sample was previously stained.
 26. Thecomposition of claim 25, wherein the biological sample was previouslystained using immunofluorescence or immunohistochemistry.
 27. Thecomposition of claim 25, wherein the biological sample was previouslystained using hematoxylin and eosin.
 28. The composition of claim 1,wherein the biological sample was previously fixed with one or both ofmethanol and acetone.
 29. The composition of claim 1, wherein thespatial array comprises one or more features.
 30. The composition ofclaim 29, wherein the one or more features comprises the plurality ofcapture probes.